EP3184547A1 - Anticorps anti-tpbg et procédés d'utilisation - Google Patents

Anticorps anti-tpbg et procédés d'utilisation Download PDF

Info

Publication number
EP3184547A1
EP3184547A1 EP15192002.2A EP15192002A EP3184547A1 EP 3184547 A1 EP3184547 A1 EP 3184547A1 EP 15192002 A EP15192002 A EP 15192002A EP 3184547 A1 EP3184547 A1 EP 3184547A1
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
tpbg
cancer
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP15192002.2A
Other languages
German (de)
English (en)
Inventor
designation of the inventor has not yet been filed The
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Priority to EP15192002.2A priority Critical patent/EP3184547A1/fr
Priority to PCT/EP2016/075881 priority patent/WO2017072207A1/fr
Priority to CN201680063211.7A priority patent/CN108347906A/zh
Priority to EP16790315.2A priority patent/EP3368576A1/fr
Priority to SG10202006332PA priority patent/SG10202006332PA/en
Priority to PCT/EP2016/075882 priority patent/WO2017072208A1/fr
Priority to SG11201802698XA priority patent/SG11201802698XA/en
Priority to CN202111178978.3A priority patent/CN113897371A/zh
Priority to JP2018522057A priority patent/JP2019500853A/ja
Priority to CN201680062256.2A priority patent/CN108350083B/zh
Priority to JP2018522020A priority patent/JP7085992B2/ja
Priority to EP16790926.6A priority patent/EP3367786A1/fr
Priority to CN202111174951.7A priority patent/CN113862300A/zh
Publication of EP3184547A1 publication Critical patent/EP3184547A1/fr
Priority to US15/965,517 priority patent/US10434184B2/en
Priority to HK19100672.4A priority patent/HK1258306A1/zh
Priority to HK19101320.8A priority patent/HK1258850A1/zh
Priority to US16/553,498 priority patent/US20200108154A1/en
Priority to US18/052,113 priority patent/US20230332175A1/en
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/462—Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • C07K16/465—Igs containing CDR-residues from one specie grafted between FR-residues from another with additional modified FR-residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to anti-TPBG antibodies and methods of using the same.
  • Trophoblast glycoprotein (TPBG) is a leucine-rich transmembrane glycoprotein involved in cell adhesion. In adults this protein is highly expressed in many tumor cells and is associated with poor clinical outcome in numerous cancers.
  • Antibodies binding to TPBG are, e.g. disclosed in Shaw et al. (2002) Biochem. J. 363: 137-45 and WO98/55607 .
  • TPBG also referred to as "5T4"
  • H8 specifically binds to a conformational epitope of TPBG.
  • Amino acid sequences of original murine H8 antibody and of a humanized version of H8 antibody are disclosed in WO 2006/031653 .
  • antibodies binding to TPGB are, e.g. disclosed and characterized in WO 2007/106744 , referred to as antibodies A1, A2 and A3.
  • Therapeutic use of antibodies binding to TPBG in treatment of cancer is, e.g., disclosed in Myers et al. (2002) Cancer Gene Ther. 9: 884-896 , Shaw et al. (2000) Biochim. Biophys. Acta. 1524: 238-246 ; and US 2003/0018004 , disclosing that anti-TPBG antibody antibody sequences fused to the human IgG1 constant domain or to the extracellular domain of murine B7.1 induces cytolysis of TPBG-expressing tumor cell lines.
  • the invention provides anti-TPBG antibodies, conjugates thereof and methods of using the same. Also provided are isolated anti-TPBG polypeptides and isolated nucleic acids encoding the same.
  • the invention relates to an isolated antibody that binds to trophoblast glycoprotein (TPBG), wherein the antibody binds to human TPBG (SEQ ID NO: 1) and to minipig TPBG (SEQ ID NO: 2).
  • TPBG trophoblast glycoprotein
  • the invention relates to an isolated antibody that binds to TPBG, wherein the antibody specifically binds to human and minipig TPBG with a KD of 10-8 M or less.
  • the invention in another aspect relates to an isolated antibody that binds to TPBG, wherein the antibody binds to human TPBG (SEQ ID NO: 1) and to minipig TPBG (SEQ ID NO: 2), and wherein the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.4.
  • the invention in another aspect relates to an isolated antibody that binds to TPBG, wherein the antibody binds to human TPBG (SEQ ID NO: 1) and to minipig TPBG (SEQ ID NO: 2), and wherein the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.4 with a KD of 10-8 M or less.
  • the invention relates to an isolated antibody that binds to TPBG, wherein the antibody binds to human TPBG (SEQ ID NO: 1) and to minipig TPBG (SEQ ID NO: 2), and wherein the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.2.
  • the antibody specifically binds to cell surface expressed human TPBG at a pH of 5.5 and a pH of 7.2.
  • the antibody specifically binds to cell surface expressed TPBG on SW620 cells at a pH of 5.5 and a pH of 7.2.
  • the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.2, wherein binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytomentry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody specifically binds to cell surface expressed human TPBG at a pH of 5.5 and a pH of 7.2, wherein binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytomentry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody specifically binds to cell surface expressed TPBG on SW620 cells at a pH of 5.5 and a pH of 7.2, wherein binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytomentry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody binds to cell surface expressed human TPBG and to cell surface expressed minipig TPBG.
  • the antibody is internalized into a human or minipig-TPBG-expressing cell upon binding to cell surface expressed TPBG.
  • the antibody comprises a CDR3 of the heavy chain of SEQ ID NO: 5, a CDR3 of the light chain of SEQ ID NO: 8, and a CDR2 of the heavy chain of SEQ ID NO: 4 (corresponding to the aforementioned CDRs of the antibody "051" as disclosed herein).
  • the antibody comprises a CDR1 of the heavy chain of SEQ ID NO: 3, a CDR2 of the heavy chain of SEQ ID NO: 4, a CDR3 of the heavy chain of SEQ ID NO: 5, a CDR1 of the light chain of SEQ ID NO: 6, a CDR2 of the light chain of SEQ ID NO: 7, and a CDR3 of the light chain of SEQ ID NO: 8 (corresponding to the six CDRs of the antibody "051" as disclosed herein).
  • the antibody comprises a CDR3 of the heavy chain of SEQ ID NO: 13, a CDR3 of the light chain of SEQ ID NO: 16, and a CDR2 of the heavy chain of SEQ ID NO: 12 (corresponding to the aforementioned CDRs of the antibody "091" as disclosed herein).
  • the antibody comprises a CDR1 of the heavy chain of SEQ ID NO: 11, a CDR2 of the heavy chain of SEQ ID NO: 12, and a CDR3 of the heavy chain of SEQ ID NO: 13, a CDR1 of the light chain of SEQ ID NO: 14, a CDR2 of the light chain of SEQ ID NO: 15, and a CDR3 of the light chain of SEQ ID NO: 16 (corresponding to the six CDRs of the antibody "091" as disclosed herein).
  • the antibody comprises a CDR3 of the heavy chain of SEQ ID NO: 21, a CDR3 of the light chain of SEQ ID NO: 24, and a CDR2 of the heavy chain of SEQ ID NO: 20 (corresponding to the aforementioned CDRs of the antibody "097" as disclosed herein).
  • the antibody comprises a CDR1 of the heavy chain of SEQ ID NO: 19, a CDR2 of the heavy chain of SEQ ID NO: 20, and a CDR3 of the heavy chain of SEQ ID NO: 21, a CDR1 of the light chain of SEQ ID NO: 22, a CDR2 of the light chain of SEQ ID NO: 23, and a CDR3 of the light chain of SEQ ID NO: 24 (corresponding to the six CDRs of the antibody "097" as disclosed herein).
  • the antibody is a humanized antibody derived from one of the aforementioned antibodies.
  • the antibody comprises
  • the invention provides an antibody comprising a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 10 (corresponding to the variable domains of the antibody "051" as disclosed herein).
  • the antibody comprises
  • the invention provides an antibody comprising a VH sequence of SEQ ID NO: 17 and a VL sequence of SEQ ID NO: 18 (corresponding to the variable domains of the antibody "091" as disclosed herein).
  • the antibody comprises
  • the invention provides an antibody comprising a VH sequence of SEQ ID NO: 25 and a VL sequence of SEQ ID NO: 26 (corresponding to the variable domains of the antibody "097" as disclosed herein).
  • the antibody is an antibody fragment that binds TPBG. In one embodiment of the invention, the antibody is a Fab fragment that binds TPBG.
  • the invention provides an isolated nucleic acid encoding the antibody of the invention. In one aspect the invention provides a host cell comprising said nucleic acid.
  • the invention provides a method of producing an antibody comprising culturing said host cell of the invention so that the antibody is produced. In one embodiment of said method the method further comprises recovering the antibody from the host cell.
  • the invention provides an immunoconjugate comprising the antibody of the invention and a cytotoxic agent.
  • the cytotoxic agent is an enzymatically active toxin or fragment thereof.
  • the cytotoxic agent is selected from the group consisting of exotoxin A chain (from Pseudomonas aeruginosa), diphtheria toxin, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • the cytotoxic agent is Pseudomonas exotoxin A or a variant thereof.
  • the invention provides a method of producing the immunoconjugate of the invention, including the step of coupling the antibody to the cytotoxic agent.
  • said method further includes the step of providing the antibody before the step of coupling, wherein the antibody is provided by culturing the host cell of the invention, which comprises a nucleic acid encoding the antibody of the invention, so that the antibody is produced.
  • said method further includes the step of recovering the antibody from the host cell before the step of coupling.
  • the invention provides a recombinant fusion protein comprising an antibody of the invention and any other polypeptide.
  • the other polypeptide is fused to at least one of the heavy chains of the antibody.
  • the other polypeptide is fused to the C-terminus of at least one of the heavy chains of the antibody.
  • the polypeptide is a cytotoxic agent (in consequence, in this embodiment, the recombinant fusion protein is an immunoconjugate).
  • the cytotoxic agent is an enzymatically active toxin or fragment thereof.
  • the cytotoxic agent is selected from the group consisting of exotoxin A chain (from Pseudomonas aeruginosa), diphtheria toxin, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • the cytotoxic agent is Pseudomonas exotoxin
  • the invention provides an isolated nucleic acid encoding the recombinant fusion protein of the invention.
  • the nucleic acid encodes the recombinant fusion protein of the invention, wherein the recombinant fusion protein is an immunoconjugate (in this embodiment, the polypeptide comprised in the recombinant fusion protein is a cytotoxic agent).
  • the invention provides a host cell comprising said nucleic acid.
  • the invention provides a method of producing a recombinant fusion protein comprising culturing said host cell so that the recombinant fusion protein is produced.
  • the recombinant fusion protein is an immunoconjugate.
  • the method further comprises recovering the recombinant fusion protein from the host cell.
  • the method further comprises isolating inclusion bodies of the host cell and solubilizing the inclusion bodies.
  • the method further comprises the step of renaturation of the material from the solubilized inclusion bodies.
  • host cell is a bacterial cell.
  • said method host cell is an Escherichia coli cell.
  • the invention provides a pharmaceutical formulation comprising the antibody of the invention and a pharmaceutically acceptable carrier.
  • said pharmaceutical composition further comprises an additional therapeutic agent.
  • said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides a pharmaceutical formulation comprising the recombinant fusion protein of the invention and a pharmaceutically acceptable carrier.
  • said pharmaceutical composition further comprises an additional therapeutic agent.
  • said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides a pharmaceutical formulation comprising the immunoconjugate of the invention and a pharmaceutically acceptable carrier.
  • said pharmaceutical composition further comprises an additional therapeutic agent.
  • said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides the antibody of the invention for use as a medicament. In one aspect the invention provides the antibody of the invention for use in the treatment of cancer.
  • the invention provides the use of the antibody of the invention in the manufacture of a medicament.
  • the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of the antibody of the invention. In one embodiment said method further comprises administering an additional therapeutic agent to the individual. In one embodiment said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides the recombinant fusion protein of the invention for use as a medicament. In one aspect the invention provides the recombinant fusion protein of the invention for use in the treatment of cancer.
  • the invention provides the use of the recombinant fusion protein of the invention in the manufacture of a medicament.
  • the medicament is for treatment of cancer
  • the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of the recombinant fusion protein of the invention. In one embodiment said method further comprises administering an additional therapeutic agent to the individual. In one embodiment said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides the immunoconjugate of the invention for use as a medicament. In one aspect the invention provides the immunoconjugate of the invention for use in the treatment of cancer.
  • the invention provides the use of the immunoconjugate of the invention in the manufacture of a medicament.
  • the medicament is for treatment of cancer.
  • the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of the immunoconjugate of the invention. In one embodiment said method further comprises administering an additional therapeutic agent to the individual. In one embodiment said additional therapeutic agent is a chemotherapeutic agent.
  • the invention provides a method of screening for an antibody that binds to human TPBG and to minipig TPBG, the method comprising the steps of testing the binding of a test antibody to human TPBG and to minipig TPBG, and selecting the antibody that binds to human TPBG and to minipig TPBG.
  • the method is for the screening of an antibody for therapeutic application.
  • the method comprises the step of testing the binding of a test antibody in monovalent form.
  • the method comprises the steps of testing the binding of a test antibody to human TPBG at pH 5.5 and at pH 7.2, and selecting the antibody that binds to human TPBG at both pH (pH 5.5 and pH 7.2).
  • the invention provides an antibody obtained by a method of screening of the invention.
  • the invention provides a method of generating an antibody that binds to human TPBG and to minipig TPBG, the method comprising the steps of (a) immunizing an animal with human TPBG or with human TBPG extracellular domain (ECD), (b) isolating B cells specific for human TPBG from the blood of the animal, (c) identifying the amino acid sequence of the variable domains (VH and VL) of the antibodies produced by the isolated B cells, (d) providing an antibody comprising the variable domains identified, (e) testing the binding of a the provided antibody to human TPBG and to minipig TPBG, and (f) selecting the antibody that binds to human TPBG and to minipig TPBG.
  • ECD extracellular domain
  • the method is for generating an antibody for therapeutic application.
  • the animal is a rodent. In one embodiment the animal is a rabbit.
  • the method comprises the step of testing the binding of the provided antibody in monovalent form.
  • the method comprises the steps of testing the binding of the provided antibody to human TPBG at pH 5.5 and at pH 7.2, and selecting the antibody that binds to human TPBG at both pH (pH 5.5 and pH 7.2).
  • the invention provides an antibody obtained by a method of generating an anti-TPBG antibody of the invention.
  • the invention provides antibodies that bind to TPBG which are cross-reactive between TPBG of human and minipig species, and thereby are suitable for clinical development as they can be used for studies in minipig species (hence avoiding necessity of monkey studies).
  • the antibodies of the invention that bind to TPBG bind to their target, i.e. human TPBG, both at neutral as well as slightly acidic pH thereby assuring antigen binding in the slightly acidic microenvironment of a tumor, which is advantageous for a therapeutic application of the antibodies in cancer therapy.
  • antibodies of the invention exhibit superior potency for treatment of several types of cancer, e.g. human non-small cell lung cancer.
  • acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • an antibody that binds to TPBG refers to an antibody that is capable of binding TPBG with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TPBG.
  • an antibody that binds to TPBG has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M).
  • an anti-TPBG antibody binds to an epitope of TPBG that is conserved among TPBG from different species.
  • test antibody refers to an antibody with unknown characteristics with respect to its binding properties to TPBG.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • an "antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • An exemplary competition assay is provided herein.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242 .
  • FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a "human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3 .
  • the subgroup is subgroup kappa I as in Kabat et al., supra .
  • the subgroup is subgroup III as in Kabat et al., supra.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions” or “CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts").
  • CDRs complementarity determining regions
  • hypervariable loops form structurally defined loops
  • antigen contacts Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • Exemplary HVRs herein include:
  • HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
  • an “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-TPBG antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3).
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • TPBG refers to any native TPBG from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses "full-length,” unprocessed TPBG as well as any form of TPBG that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TPBG, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human TPBG is shown in SEQ ID NO: 1.
  • the amino acid sequence of an exemplary minipig TPBG is shown in SEQ ID NO: 2.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887 ; Clackson, T. et al., Nature 352 (1991) 624-628 ).
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • Cancer as used herein include both solid and haematologic cancers, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the
  • Pseudomonas exotoxin A Native, wild-type Pseudomonas exotoxin A is a 66kD bacterial toxin secreted by Pseudomonas aeruginosa, having the 613 amino acid sequence shown in SEQ ID NO:52 and also disclosed in US 5,602,095 . This sequence is shown without the native signal peptide, which is shown as the first 25 amino acids of UniProt accession number P11439.2 (gi: 12231043).
  • the native protein has three major structural domains.
  • the N-terminal domain I comprises two subdomains Ia (amino acids 1-252) and Ib (amino acids 365-399) that are structurally adjacent but separated in the primary amino acid sequence.
  • Domain I and in particular domain Ia is the cell-binding domain.
  • the function of domain Ib remains undefined.
  • Domain I forms the major component of the B subunit.
  • forms of PE in which the native domain Ia sequence is omitted or disrupted, and which consequently are unable to bind to LRP1 or LRP1B, are greatly preferred.
  • Domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2.
  • the structural boundary between domain Ib and domain III is not fully settled. According to WO2013/040141 it lies between residues 399 and 400, but Weldon and Pastan 2011 place it between residues 404 and 405.
  • full catalytic activity requires a portion of domain Ib as well as domain III.
  • the functional domain III of the native toxin is defined to start at residue 395.
  • Amino acids 602-613 have been found to be inessential for NAD(+)-ribosyltransferase activity, but amino acids 609-613 of the native sequence are required for cytotoxic activity.
  • Pseudomonas exotoxin A is specifically intended to include the variants and improvements disclosed in WO88/02401A1 , WO90/12592A1 , WO91/09949 , WO91/09965 , WO93/25690 , WO97/13529 , WO98/20135 , WO2005/052006 , WO2007/016150 , WO2007/031741 , WO2009/32954 , WO2011/32022 , WO2012/154530 , WO2012/170617 , WO2013/40141 , Mazor R, et al PNAS 111 (2014) 8571-8576 , and Alewine C, et al, Mol Cancer Ther.
  • a PE toxin will have a polypeptide sequence comprising a PE functional domain III having at least 50% amino acid sequence identity over the full length of residues 395-601 of SEQ ID NO: 52, wherein the PE toxin has cytotoxic activity when introduced into a eukaryotic (preferably mammalian) cell.
  • Preferred forms of PE comprise (1) a PE functional domain III having at least 50% amino acid sequence identity over the full length of residues 395-601 of SEQ ID NO: 52 and having NAD(+)-diphthamide ADP ribosyltransferase activity, and (2) at least one endoplasmic reticulum localisation sequence.
  • the PE in embodiments in which the PE is coupled to a cell-binding agent as a fusion polypeptide, the PE preferably also comprises (3) a cleavable linker sequence such as a furin-cleavable sequence (FCS) that permits cleavage of the PE functional domain III from the cell-binding agent following uptake into the target cell.
  • a cleavable linker sequence such as a furin-cleavable sequence (FCS) that permits cleavage of the PE functional domain III from the cell-binding agent following uptake into the target cell.
  • FCS furin-cleavable sequence
  • the cleavable linker (such as an FCS) will generally be on the N-terminal side of the PE functional domain III.
  • cleavable linkers may be used provided that they permit cleavage of the PE from the cell-binding agent following uptake into the target cell.
  • other means of coupling the PE to the cell-binding agent are contemplated, provided again that they permit separation of the PE from the cell-binding agent following uptake into the target cell.
  • the cell-binding agent may be non-covalently linked to the PE, or linked by disulfide bonds which permit release of the PE moiety under reducing conditions, or linked by other conjugation chemistries that are known in the field of immunoconjugate production.
  • the PE for use in accordance with the present invention will generally lack a functional cell-binding domain I.
  • PE40 is a truncated derivative of PE ( Pai et al 1991 Proc. Natl. Acad. Sci. USA 88:3358-62 and Kondo et al. 1988 J. Biol. Chem. 263:9470-9475 ).
  • PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1-279 have been deleted and the molecule commences with a Met at position 280 followed by amino acids 281-364 and 381-613 of PE as defined by reference to SEQ ID NO: 52.
  • PE35 and PE40 are disclosed, for example, in US 5,602,095 , US 4,892,827 , WO93/25690 and WO88/02401 , each of which is incorporated herein by reference in its entirety.
  • PE38 contains the translocating and ADP ribosylating domains of PE but not the cell-binding portion ( Hwang et al. 1987 Cell 48:129-136 ).
  • PE38 (SEQ ID NO: 53) is a truncated PE pro-protein composed of amino acids 253-364 and 381-613 of SEQ ID NO: 52 which is activated to its cytotoxic form upon processing within a cell (see US 5,608,039 , which is incorporated by reference in its entirety herein, and Pastan et al. 1997 Biochim. Biophys. Acta, 1333:C1-C6 ).
  • PE38QR is a variant of PE38 having mutations of the lysines at positions 590, 606 and 613 of domain III, to permit conjugation to antibodies.
  • PE-LR contains a deletion of domain II except for a furin-cleavable sequence (FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 52 (RHRQPRGWEQL (SEQ ID NO: 54)) and a deletion of amino acid residues 365-394 of domain lb.
  • FCS furin-cleavable sequence
  • PE-LR contains amino acid residues 274-284 and 395-613 of SEQ ID NO: 52.
  • PE-LR is described in WO 2009/032954 and Weldon et al 2009 Blood 113:3792-3800 , which are each incorporated herein by reference in their entirety.
  • WO2012/154530 describes that the addition of a short, flexible linker of between 3 and 8 amino acids each independently selected from glycine and serine between the FCS and the PE functional domain III improves the cytotoxicity of the PE-LR molecule without disrupting binding by furin.
  • exemplary linkers are GGS and GGSGGS (SEQ ID NO: 55).
  • WO2012/154530 reports that substitutions at the following amino acid residues within PE domain III reduce immunogenicity:
  • Preferred substitutions are with a glycine, serine or alanine residue.
  • WO2012/170617 reports that substitutions at these residues may reduce immunogenicity of B cell epitopes, and that substitutions at one or more of residues R427, R458, R467, R490, R505 and F538 are preferred, particularly with alanine.
  • WO2013/040141 reports that substitutions at the following additional amino acid residues may reduce the immunogenicity of B cell epitopes within PE domain III:
  • Preferred substitutions are with a glycine, serine, alanine or glutamine residue.
  • WO2012/170617 reports that substitutions at the following residues can reduce the immunogenicity of T-cell epitopes within PE domain III:
  • Preferred substitutions are at one or more of residues D463, Y481 and L516, which may also reduce the immunogenicity of B cell epitopes.
  • Preferred substitutions are with a glycine, serine, alanine or glutamine residue.
  • WO2012/170617 also reports that substitutions at the following amino acid residues can reduce the immunogenicity of T cell epitopes within PE domain II:
  • Preferred substitutions are with a glycine, serine, alanine or glutamine residue.
  • WO2012/170617 also reports that substitutions at the following amino acid residues can reduce the immunogenicity of B cell epitopes within PE domain II:
  • WO2012/170617 also reports that a particularly preferred combination of substitutions is D463A/R427A/R458A/R467A/R490A/R505A/R538A.
  • the PE functional domain III may comprise mutations at any one or any combination of more than one of the following sites:
  • the mutation(s) reduce(s) the immunogenicity compared to the unmutated sequence of the amino acids 395-613 of SEQ ID NO: 52.
  • the PE contains some or all of domain II, it may comprise mutations at any one or any combination of more than one of the following sites:
  • the mutation(s) reduce(s) the immunogenicity compared to the unmutated sequence from domain II.
  • the FCS is derived from the native furin-cleavable sequence of PE consisting of amino acids 274-284 (RHRQPRGWEQL, SEQ ID NO: 54) may comprise a substitution of the E282 residue, especially if the adjacent sequence from the native PE sequence is included downstream of the FCS.
  • the adjacent sequence from the native PE sequence is not included (such as PE-LR, in which the FCS is fused to domain III either directly or via a non-native linker sequence)
  • the epitope from the native sequence may anyway be disrupted such that a mutation at the E282 residue may not be advantageous.
  • Reduced immunogenicity in variant PE toxins may refer to a reduced ability of the variant sequence to induce a T cell response and/or a reduced ability of the variant sequence to induce a B cell (antibody) response, preferably both.
  • Techniques for assessing the effect of mutations on T cell immunogenicity are well known in the art and described in the examples of WO 2012/170617 .
  • Techniques for assessing the effect of mutations on the B cell immunogenicity are likewise well known in the art and described in WO 2013/040141 , for example.
  • Human antibodies may be raised against the native PE sequence by phage display using a human antibody library. The ability of mutations in the PE sequence to disrupt binding of such antibodies to the variant PE molecule is indicative of reduced immunogenicity.
  • the titre of PE-specific antibodies raised in transgenic mice carrying the human antibody repertoire may be compared for the native and mutated PE sequences.
  • the C-terminal end of the PE functional domain III may contain the native sequence of residues 609-613, namely REDLK (SEQ ID NO: 55). Additionally or alternatively to any other modifications of the native PE sequence, the PE functional domain III may contain a variant of the REDLK sequence, or other sequences, that function to maintain the PR protein in the endoplasmic reticulum or to recycle proteins into the endoplasmic reticulum. Such sequences are referred to here as "endoplasmic reticulum localisation sequences" or "ER localisation sequences”.
  • Preferred ER localisation sequences include such as KDEL (SEQ ID NO: 57), REDL (SEQ ID NO: 58), RDEL (SEQ ID NO: 59) or KEDLK (SEQ ID NO: 60).
  • KDEL SEQ ID NO: 57
  • REDL SEQ ID NO: 58
  • RDEL SEQ ID NO: 59
  • KEDLK SEQ ID NO: 60
  • One or more additional ER localisation sequences may be added to the C-terminal end of the PE polypeptide sequence.
  • KDEL, or 2 or 3 tandem repeats of KDEL (KDELKDEL, SEQ ID NO 61; KDELKDELKDEL, SEQ ID NO: 62) for the native REDLK sequence, or the addition of KDEL after the native REDLK sequence is preferred. See for example WO91/099949 , Chaudhary et al 1991 Seetharam et al 1991.
  • WO91/09949 discloses that the C-terminal end of the PE functional domain III may lack some or all of residues 602-608, which are not essential for the NAD(+)-diphthamide ADP ribosyltransferase activity.
  • Furin-cleavable sequence can be any polypeptide sequence cleavable by furin.
  • Duckert et al. 2004, Protein Engineering, Design & Selection 17(1):107-112 (hereafter, "Duckert et al.") is incorporated herein by reference in its entirety and particularly with regard to the furin-cleavable sequences and motifs it discloses.
  • Duckert et al. discloses that furin is an enzyme in a family of evolutionarily conserved dibasic-and monobasic-specific CA2+-dependent serine proteases called substilisin/kexin-like proprotein convertases. See page 107.
  • Furin also known as "paired basic amino acid cleaving enzyme", "PACE", or PCSK3
  • PACE paired basic amino acid cleaving enzyme
  • PCSK3 paired basic amino acid cleaving enzyme
  • the minimal furin-cleavable sequence typically is, in the single letter code for amino acid residues, R-X-X-R (SEQ ID NO: 63), with cleavage occurring after the second "R".
  • Duckert et al. summarizes the information available on the sequences of 38 proteins reported in the literature to have furin-cleavable sequences, including mammalian proteins, proteins of pathogenic bacteria, and viral proteins. It reports that 31, or 81%, of the cleavage motifs reviewed had the R-X-[R/K]-R (SEQ ID NO: 64, SEQ ID NO.
  • cleavage motifs contained only the minimal cleavage sequence.
  • Duckert et al. further aligned the motifs and identified the residues found at each position in each furin both for the cleavage motif itself and in the surrounding residues.
  • Fig. 1A of Duckert et al. shows by relative size the residues most commonly found at each position. By convention, the residues surrounding the furin cleavage site are numbered from the scissile bond (which is typically indicated by a downward arrow).
  • the substrate residues are designated P1, P2, and so on, while counting towards the C-terminus, the residues are designated P1', P2', and so on.
  • the residues are designated P1', P2', and so on.
  • furin cleavage occurs between arginine 279 and glycine 280 in an arginine-rich loop located in domain II of the toxin.
  • the native furin-cleavable sequence in domain II of PE is set forth below (with numbers indicating the positions of the residues in the 613-amino acid native PE sequence), and aligned to show its numbering under the convention noted above:
  • This sequence has shown a cleavage rate faster than that of the native sequence, and when used in an exemplary immunotoxin resulted in cytotoxicity to target cells approximately the same as that of the native sequence.
  • a furin-cleavable sequence used to attach the targeting molecule to PE domain III can be the minimal furin-cleavable sequence, R-X-X-R (wherein each X is independently any naturally occurring amino acid), preferably R-X-[R/K]-R (wherein X is any naturally occurring amino acid and [R/K] denotes either arginine or lysine), or any of the other furin-cleavable sequences known in the art or permitted by Fig.
  • the sequence can be RKKR (SEQ ID NO: 68), RRRR (SEQ ID NO: 69), RKAR (SEQ ID NO: 70), SRVARS (SEQ ID NO: 71), TSSRKRRFW (SEQ ID NO: 72), or ASRRKARSW (SEQ ID NO: 73).
  • the furin-cleavable sequence is RRVKKRFW (SEQ ID NO: 74), RNVVRRDW (SEQ ID NO: 75), or TRAVRRRSW (SEQ ID NO: 76).
  • the residue at position PI can be the arginine present in the native sequence, or lysine.
  • a lysine can be substituted for the arginine at position PI in, for example, any the sequences set forth above.
  • the furin-cleavable sequence contains the native furin-cleavable sequence of PE: R-H-R-Q-P-R-G-W-E-Q-L (SEQ ID NO: 66) or a truncated version of the native sequence, so long as it contains the minimal furin-cleavable sequence and is cleavable by furin.
  • the furin-cleavable sequence can be RQPR (SEQ ID NO: 77), RHRQPRGW (SEQ ID NO: 78), RHRQPRGWE (SEQ ID NO: 79), HRQPRGWEQ (SEQ ID NO: 80), or RQPRGWE (SEQ ID NO: 81).
  • the sequence is RHRSKRGWEQL (SEQ ID NO: 67) or a truncated version of this sequence, so long as it contains the minimal furin-cleavable sequence and is cleavable by furin.
  • the furin-cleavable sequence can be RSKR (SEQ ID NO: 82), RHRSKRGW (SEQ ID NO: 83), HRSKRGWE (SEQ ID NO: 84), RSKRGWEQL (SEQ ID NO: 85), HRSKRGWEQL (SEQ ID NO: 86), or RHRSKR (SEQ ID NO: 87).
  • the E282 residue at the P3' position of FCS sequences derived from PE may be replaced by another amino acid to reduce B cell immunogenicity. Where the sequence lacks native PE residues downstream of this residue, or where the FCS contains other mutations relative to the native PE sequence, such replacement may not be necessary.
  • Whether or not any particular sequence is cleavable by furin can be determined by methods known in the art. For example, whether or not a sequence is cleavable by furin can be tested by incubating the sequence with furin in furin buffer (0.2 M NaOAc (pH 5.5), 5 mM CaCl2) at a 1 :10 enzyme:substrate molar ratio at 25°C for 16 hours. These conditions have previously been established as optimal for furin cleavage of PE.
  • the furin used is human furin. Recombinant truncated human furin is commercially available, for example, from New England Biolabs (Beverly, MA). See also, Bravo et al. 1994 J Biol Chem 269(14):25830-25837 .
  • a furin-cleavable sequence can be tested by making it into an immunotoxin with an antibody against a cell surface protein and testing the resulting immunotoxin on a cell line expressing that cell surface protein.
  • Suitable antibody sequences are disclosed in, for example, WO2012/154530 and WO2009/032954 .
  • Preferred PE toxins for use in accordance with the present invention have the following structure:
  • FCS is a furin-cleavable sequence, preferably (i) R-H-R-Q-P-R-G-W-E-Q-L or a truncated version thereof containing R-Q-P-R, optionally R-Q-P-R, R-H-R-Q-P-R-G-W, R-H-R-Q-P-R-G-W-E, H-R-Q-P-R-G-W-E-Q, or R-Q-P-R-G-W-E; or (ii) R-H-R-S-K-R-G-W-E-Q-L or a truncated version thereof containing R-S-K-R, optionally R-S-K-R, R-H-R-S-K-R-G-W, H-R-S-K-R-G-W-E, R-S-K-R-G-WE-Q-L, H-R-S-K-R-G-W
  • PE toxins for use in accordance with the present invention comprise the amino acid sequence corresponding to amino acid residues 395-613 of SEQ ID NO: 52 with Ala substitutions at positions 427, 456, 463, 467, 490, 505 and 538 (as disclosed in WO2015/51199 as LO10R-456A and SEQ ID NO:37) or the amino acid sequence corresponding to amino acid residues 395-613 of SEQ ID NO: 52 with Ala substitutions at positions 427, 443, 477, 494, 505 and 552 (as is disclosed in WO2015/051199 as T18/T20 and SEQ ID NO:289).
  • the invention is based, in part, on antibodies that are crossreactive with human and minipig TPGF.
  • antibodies that bind to human TPBG at neutral as well as at slightly acidic pH are provided.
  • Antibodies of the invention are useful, e.g., for the diagnosis or treatment of cancer.
  • the invention provides isolated antibodies that bind to TPBG.
  • an anti-TPBG antibody of the invention binds to human TPBG according to SEQ ID NO: 1 and binds to to minipig TPBG according to SEQ ID NO: 2.
  • an anti-TPBG antibody of the invention binds to human TPBG at a pH of 5.5 and at a pH of 7.2.
  • the invention provides an anti-TPBG antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR1 of the heavy chain of SEQ ID NO: 3, (b) CDR2 of the heavy chain of SEQ ID NO: 4, (c) CDR3 of the heavy chain of SEQ ID NO: 5, (d) CDR1 of the light chain of SEQ ID NO: 6, (e) CDR2 of the light chain of SEQ ID NO: 7, and (f) CDR3 of the light chain of SEQ ID NO: 8 (corresponding to the aforementioned CDRs of the antibody "051" as disclosed herein).
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR1 of the heavy chain of SEQ ID NO: 3, (b) CDR2 of the heavy chain of SEQ ID NO: 4, (c) CDR3 of the heavy chain of SEQ ID NO: 5 (corresponding to the aforementioned CDRs of the antibody "051" as disclosed herein).
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 5.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 5, CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 5, and CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 4.
  • the antibody comprises (a) CDR3 of the heavy chain of SEQ ID NO: 5, (b) CDR3 of the light chain of SEQ ID NO: 8, and (c) CDR2 of the heavy chain of SEQ ID NO: 4.
  • the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 6; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 7; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the antibody comprises (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 6; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 7; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 4, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 5; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 6, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 7, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the invention provides an antibody comprising (a) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3, (b) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 4, and (c) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 5, (d) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 6, (e) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 7, and (f) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the anti-TPBG antibody is a humanized antibody derived from an antibody comprising the aforementioned CDRs.
  • the invention provides an anti-TPBG antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR1 of the heavy chain of SEQ ID NO: 11, (b) CDR2 of the heavy chain of SEQ ID NO: 12, (c) CDR3 of the heavy chain of SEQ ID NO: 13, (d) CDR1 of the light chain of SEQ ID NO: 14, (e) CDR2 of the light chain of SEQ ID NO: 15, and (f) CDR3 of the light chain of SEQ ID NO: 16 (corresponding to the aforementioned CDRs of the antibody "091" as disclosed herein).
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR1 of the heavy chain of SEQ ID NO: 11, (b) CDR2 of the heavy chain of SEQ ID NO: 12, (c) CDR3 of the heavy chain of SEQ ID NO: 13 (corresponding to the aforementioned CDRs of the antibody "091" as disclosed herein).
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 13.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 13, CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 13, and CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 12.
  • the antibody comprises (a) CDR3 of the heavy chain of SEQ ID NO: 13, (b) CDR3 of the light chain of SEQ ID NO: 16, and (c) CDR2 of the heavy chain of SEQ ID NO: 12.
  • the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 14; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 15; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • the antibody comprises (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 14; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 15; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 11, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 13; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 14, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 15, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • the invention provides an antibody comprising (a) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 11, (b) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and (c) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 13, (d) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 14, (e) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 15, and (f) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • the anti-TPBG antibody is a humanized antibody derived from an antibody comprising the aforementioned CDRs.
  • the invention provides an anti-TPBG antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR1 of the heavy chain of SEQ ID NO: 19, (b) CDR2 of the heavy chain of SEQ ID NO: 20, (c) CDR3 of the heavy chain of SEQ ID NO: 21, (d) CDR1 of the light chain of SEQ ID NO: 22, (e) CDR2 of the light chain of SEQ ID NO: 23, and (f) CDR3 of the light chain of SEQ ID NO: 24 (corresponding to the aforementioned CDRs of the antibody "097" as disclosed herein).
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR1 of the heavy chain of SEQ ID NO: 19, (b) CDR2 of the heavy chain of SEQ ID NO: 20, (c) CDR3 of the heavy chain of SEQ ID NO: 21 (corresponding to the aforementioned CDRs of the antibody "097" as disclosed herein).
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • the antibody comprises CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21, CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 21, and CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 20.
  • the antibody comprises (a) CDR3 of the heavy chain of SEQ ID NO: 21, (b) CDR3 of the light chain of SEQ ID NO: 24, and (c) CDR2 of the heavy chain of SEQ ID NO: 20.
  • the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 22; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 23; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • the antibody comprises (a) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 22; (b) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 23; and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 21; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 23, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • the invention provides an antibody comprising (a) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 19, (b) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and (c) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 21, (d) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 22, (e) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 23, and (f) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • the anti-TPBG antibody is a humanized antibody derived from an antibody comprising the aforementioned CDRs.
  • an anti-TPBG antibody is humanized.
  • an anti-TPBG antibody comprises CDRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
  • an anti-TPBG antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9 (corresponding to the variable heavy chain domain of the antibody "051" as disclosed herein).
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to TPBG.
  • the anti-TPBG antibody comprises the VH sequence in SEQ ID NO: 9, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 3, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 4, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 5.
  • an anti-TPBG antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to PRO.
  • the anti-TPBG antibody comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence.
  • the VL comprises one, two or three HVRs selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 6, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 7, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • an anti-TPBG antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 9 and SEQ ID NO: 10, respectively, including post-translational modifications of those sequences.
  • the invention provides an antibody that binds to the same epitope as an anti-TPBG antibody provided herein.
  • an antibody is provided that binds to the same epitope as an anti-TPBG antibody comprising a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 10.
  • an anti-TPBG antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17 (corresponding to the variable heavy chain domain of the antibody "091" as disclosed herein).
  • VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to TPBG.
  • the anti-TPBG antibody comprises the VH sequence in SEQ ID NO: 17, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 11, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 13.
  • an anti-TPBG antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to PRO.
  • the anti-TPBG antibody comprises the VL sequence in SEQ ID NO: 18, including post-translational modifications of that sequence.
  • the VL comprises one, two or three HVRs selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 14, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 15, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16.
  • an anti-TPBG antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of those sequences.
  • the invention provides an antibody that binds to the same epitope as an anti-TPBG antibody provided herein.
  • an antibody is provided that binds to the same epitope as an anti-TPBG antibody comprising a VH sequence of SEQ ID NO: 17 and a VL sequence of SEQ ID NO: 18.
  • an anti-TPBG antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25 (corresponding to the variable heavy chain domain of the antibody "097" as disclosed herein).
  • VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to TPBG.
  • the anti-TPBG antibody comprises the VH sequence in SEQ ID NO: 25, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (i) CDR1 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR2 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR3 of the heavy chain comprising an amino acid sequence selected from SEQ ID NO: 21.
  • an anti-TPBG antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TPBG antibody comprising that sequence retains the ability to bind to PRO.
  • the anti-TPBG antibody comprises the VL sequence in SEQ ID NO: 26, including post-translational modifications of that sequence.
  • the VL comprises one, two or three HVRs selected from (i) CDR1 of the light chain comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR2 of the light chain comprising the amino acid sequence of SEQ ID NO: 23, and (c) CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • an anti-TPBG antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 25 and SEQ ID NO: 26, respectively, including post-translational modifications of those sequences.
  • the invention provides an antibody that binds to the same epitope as an anti-TPBG antibody provided herein.
  • an antibody is provided that binds to the same epitope as an anti-TPBG antibody comprising a VH sequence of SEQ ID NO: 17 and a VL sequence of SEQ ID NO: 18.
  • an anti-TPBG antibody is a monoclonal antibody, including a chimeric, humanized or human antibody.
  • an anti-TPBG antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') 2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgG1 antibody or other antibody class or isotype as defined herein.
  • the invention relates to an isolated antibody that binds to TPBG, wherein the antibody binds to human TPBG (SEQ ID NO: 1) and to minipig TPBG (SEQ ID NO: 2), and wherein the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.2.
  • the antibody specifically binds to cell surface expressed human TPBG at a pH of 5.5 and a pH of 7.2.
  • the antibody specifically binds to cell surface expressed TPBG on SW620 cells at a pH of 5.5 and a pH of 7.2.
  • binding ratio is herein defined as the ratio of the mean fluorescence intensity detected by flow cytometry in a cell surface binding assay (in one embodiment an assay as described in Example 18) at pH 7.2 to the mean fluorescence intensity detected by flow cytometry at pH 5.5 (in one embodiment an assay as described in Example 18).
  • the antibody specifically binds to human TPBG at a pH of 5.5 and a pH of 7.2, wherein the binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytometry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody specifically binds to cell surface expressed human TPBG at a pH of 5.5 and a pH of 7.2, wherein the binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytometry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody specifically binds to cell surface expressed human TPBG at a pH of 5.5 and a pH of 7.2, wherein a binding ratio, which is defined as the ratio of the mean fluorescence intensity detected by flow cytometry in a cell surface binding assay (in one embodiment an assay as described in Example 18) at pH 7.2 to the mean fluorescence intensity detected by flow cytometry at pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • a binding ratio which is defined as the ratio of the mean fluorescence intensity detected by flow cytometry in a cell surface binding assay (in one embodiment an assay as described in Example 18) at pH 7.2 to the mean fluorescence intensity detected by flow cytometry at pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the antibody specifically binds to cell surface expressed TPBG on SW620 cells at a pH of 5.5 and a pH of 7.2, wherein binding ratio at pH 7.2 to pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • the binding is detected via flow cytometry and the binding ratio is determined by comparing mean fluorescence intensities as described in Example 18.
  • the antibody specifically binds to cell surface expressed TPBG on SW620 cells at a pH of 5.5 and a pH of 7.2, wherein a binding ratio, which is defined as the ratio of the mean fluorescence intensity detected by flow cytometry in a cell surface binding assay (in one embodiment an assay as described in Example 18) at pH 7.2 to the mean fluorescence intensity detected by flow cytometry at pH 5.5 is less than 1.5, in one preferred embodiment between 0.8 and 1.5, in another preferred embodiment between 0.9 and 1.2.
  • an anti-TPBG antibody may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:
  • an antibody provided herein has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M).
  • Kd dissociation constant
  • Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
  • Solution binding affinity of FABs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881 ).
  • MICROTITER ® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).
  • a non-adsorbent plate (Nunc #269620)
  • 100 pM or 26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta, L.G.
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20 ® ) in PBS. When the plates have dried, 150 ⁇ l/well of scintillant (MICROSCINT-20TM; Packard) is added, and the plates are counted on a TOPCOUNTTM gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
  • Kd is measured using surface plasmon resonance assays using a BIACORE ® -2000 or a BIACORE ® -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ⁇ 10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N -ethyl- N' - (3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N -hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml ( ⁇ 0.2 ⁇ M) before injection at a flow rate of 5 ⁇ l/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of approximately 25 ⁇ l/min.
  • TWEEN-20TM polysorbate 20
  • association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio k off /k on. See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881 .
  • an antibody provided herein is an antibody fragment.
  • Antibody fragments include, but are not limited to Fab, Fab', Fab'-SH, F(ab') 2 , Fv, and scFv fragments, and other fragments described below.
  • Fab fragment-specific antibody fragment
  • Fab' fragment-specific Fab
  • Fab'-SH fragment-specific Fab
  • F(ab') 2 fragment-specific Fab
  • Fv fragment antigen Vpent antibody fragments
  • scFv fragments see, e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 0 404 097 ; WO 1993/01161 ; Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134 ; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448 . Triabodies and tetrabodies are also described in Hudson, P.J. et al., Nat. Med. 9 (20039 129-134 ).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 ).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • recombinant host cells e.g. E. coli or phage
  • an antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567 ; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855 ).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308 ; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289 ; and Presta, L.G. et al., J. Immunol.
  • an antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459 .
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125 See also, e.g., U.S. Patent Nos.
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005 ; Brodeur, B.R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63 ; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95 ) Human antibodies generated via human B-cell hybridoma technology are also described in Li, J. et al., Proc. Natl.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
  • Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom, H.R. et al., Methods in Molecular Biology 178 (2001) 1-37 and further described, e.g., in the McCafferty, J. et al., Nature 348 (1990) 552-554 ; Clackson, T. et al., Nature 352 (1991) 624-628 ; Marks, J.D. et al., J. Mol. Biol.
  • repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455 .
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • scFv single-chain Fv
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths, A.D. et al., EMBO J. 12 (1993) 725-734 .
  • naive libraries can also be made synthetically by cloning non-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388 .
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373 , and US Patent Publication Nos. 2005/0079574 , 2005/0119455 , 2005/0266000 , 2007/0117126 , 2007/0160598 , 2007/0237764 , 2007/0292936 , and 2009/0002360 .
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
  • an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
  • one of the binding specificities is for TPBG and the other is for any other antigen.
  • bispecific antibodies may bind to two different epitopes of TPBG.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TPBG.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540 , WO 93/08829 , and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659 ), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168 ).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules ( WO 2009/089004 ); crosslinking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980 , and Brennan, M. et al., Science 229 (1985) 81-83 ); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553 ; using "diabody” technology for making bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad.
  • the antibody or fragment herein also includes a "Dual Acting Fab” or “DAF” comprising an antigen binding site that binds to TPBG as well as another, different antigen (see, US 2008/0069820 , for example).
  • the antibody or fragment herein also includes multispecific antibodies described in WO 2009/080251 , WO 2009/080252 , WO 2009/080253 , WO 2009/080254 , WO 2010/112193 , WO 2010/115589 , WO 2010/136172 , WO 2010/145792 , and WO 2010/145793 .
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Exemplary changes are provided in the following Table under the heading of "exemplary substitutions", and as further described below in reference to amino acid side chain classes. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions”.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g . a humanized or human antibody).
  • a parent antibody e.g . a humanized or human antibody
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196 ), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • HVR "hotspots” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196 ), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described,
  • variable genes chosen for maturation are introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR "hotspots" or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham, B.C. and Wells, J.A., Science 244 (1989) 1081-1085 .
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32 .
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546 , for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US 2003/0157108 ; US 2004/0093621 .
  • Examples of publications related to "defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108 ; WO 2000/61739 ; WO 2001/29246 ; US 2003/0115614 ; US 2002/0164328 ; US 2004/0093621 ; US 2004/0132140 ; US 2004/0110704 ; US 2004/0110282 ; US 2004/0109865 ; WO 2003/085119 ; WO 2003/084570 ; WO 2005/035586 ; WO 2005/035778 ; WO 2005/053742 ; WO 2002/031140 ; Okazaki, A. et al., J. Mol. Biol.
  • Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation ( Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545 ; US 2003/0157108 ; and WO 2004/056312 , especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622 ; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688 ; and WO 2003/085107 ).
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 ; US Patent No. 6,602,684 ; and US 2005/0123546 . Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 ; WO 1998/58964 ; and WO 1999/22764 .
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g ., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g . a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492 .
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063 ; and Hellstrom, I.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656 .
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402 .
  • a CDC assay may be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol.
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int. Immunol. 18 (2006: 1759-1769 ).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 ( U.S. Patent No. 6,737,056 ).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine ( US Patent No. 7,332,581 ).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e ., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642 , and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184 .
  • CDC Complement Dependent Cytotoxicity
  • Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US 2005/0014934 .
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 ( US Patent No. 7,371,826 ).
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541 .
  • an antibody provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., gly
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the non-proteinaceous moiety is a carbon nanotube ( Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605 ).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567 .
  • isolated nucleic acid encoding an anti-TPBG antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of making an anti-TPBG antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., US 5,648,237 , US 5,789,199 , and US 5,840,523 . (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254 , describing expression of antibody fragments in E. coli .)
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414 ; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215 .
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177 , 6,040,498 , 6,420,548 , 7,125,978 , and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74 ); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68 ; MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells ( Urlaub, G. et al., Proc. Natl.
  • myeloma cell lines such as Y0, NS0 and Sp2/0.
  • myeloma cell lines suitable for antibody production see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268 .
  • Anti-TPBG antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
  • an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
  • competition assays may be used to identify an antibody that competes with an antibody selected from the group of antibodies 051, 091, or 097 as disclosed herein and defined by their respective amino acid sequences of VH and VL domains for binding to TPBG.
  • a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an antibody selected from the group of antibodies 051, 091, or 097 as disclosed herein and defined by their respective amino acid sequences of VH and VL domains.
  • epitope e.g., a linear or a conformational epitope
  • Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris, G.E. (ed.), Epitope Mapping Protocols, In: Methods in Molecular Biology, Vol. 66, Humana Press, Totowa, NJ (1996 ).
  • immobilized TPBG is incubated in a solution comprising a first labeled antibody that binds to TPBG (e.g., an antibody selected from the group of antibodies 051, 091, or 097 as disclosed herein and defined by their respective amino acid sequences of VH and VL domains) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to TPBG.
  • the second antibody may be present in a hybridoma supernatant.
  • immobilized TPBG is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody.
  • assays are provided for identifying anti-TPBG antibodies thereof having biological activity.
  • Biological activity may include, e.g., binding to TPBG-expressing tumor cells.
  • Antibodies having such biological activity in vivo and/or in vitro are also provided.
  • an antibody of the invention is tested for such biological activity.
  • the invention also provides immunoconjugates comprising an anti-TPBG antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see US 5,208,020 , US 5,416,064 and EP 0 425 235 B1 ); an auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US 5,635,483 , US 5,780,588 , and US 7,498,298 ); a dolastatin; a calicheamicin or derivative thereof (see US 5,712,374 , US 5,714,586 , US 5,739,116 , US 5,767,285 , US 5,770,701 , US 5,770,710 , US 5,773,001 , and US 5,877,296 ; Hinman, L.M.
  • ADC antibody-drug conjugate
  • an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of exotoxin A chain (from Pseudomonas aeruginosa), diphtheria toxin, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • the enzymatically active toxin is Pseudomonas exotoxin A or a variant thereof.
  • an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a variety of radioactive isotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example TC 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as
  • a ricin immunotoxin can be prepared as described in Vitetta, E.S. et al., Science 238 (1987) 1098-1104 .
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026 .
  • the linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker Chari, R.V. et al., Cancer Res. 52 (1992) 127-131 ; U.S. Patent No. 5,208,020 ) may be used.
  • immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to,
  • BMPS BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,
  • SMCC SMPB
  • SMPH sulfo-EMCS
  • sulfo-GMBS sulfo-KMUS
  • sulfo-MBS sulfo-SIAB
  • sulfo-SMCC and sulfo-SMPB
  • SVSB succinimidyl-(4-vinylsulfone)benzoate
  • Conjugates of the antibody and cytotoxic agents which are polypeptides, may also be provided as recombinant fusion proteins, e.g. by direct expression of said fusion protein within a host cell, thereby not requiring an additional coupling step for provision.
  • any of the anti-TPBG antibodies provided herein is useful for detecting the presence of TPBG in a biological sample.
  • the term "detecting" as used herein encompasses quantitative or qualitative detection.
  • a biological sample comprises a cell or tissue, such as tumor cells.
  • an anti-TPBG antibody for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of TPBG in a biological sample comprises contacting the biological sample with an anti-TPBG antibody as described herein under conditions permissive for binding of the anti-TPBG antibody to TPBG, and detecting whether a complex is formed between the anti-TPBG antibody and TPBG.
  • Such method may be an in vitro or in vivo method.
  • an anti-TPBG antibody is used to select subjects eligible for therapy with an anti-TPBG antibody, e.g. where TPBG is a biomarker for selection of patients.
  • Exemplary disorders that may be diagnosed using an antibody of the invention include cancer, e.g. lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of
  • labeled anti-TPBG antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase ( U.S. Patent No.
  • luciferin 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • HRP horseradish peroxidase
  • lactoperoxidase lactoperoxidase
  • microperoxidase biotin/avidin
  • spin labels bacteriophage labels,
  • compositions of an anti-TPBG antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980 )), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rhuPH20 HYLENEX ® , Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968 .
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958 .
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908 , the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients preferably those with complementary activities that do not adversely affect each other.
  • chemotherapeutic agent preferably a chemotherapeutic agent listed below.
  • Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g . films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • any of the anti-TPBG antibodies provided herein, or the recombinant fusion proteins or immunoconjugates comprising the anti-TPBG antibodies provided herein, may be used in therapeutic methods.
  • an anti-TPBG antibody for use as a medicament is provided.
  • an anti-TPBG antibody for use in treating cancer is provided.
  • an anti-TPBG antibody for use in a method of treatment is provided.
  • the invention provides an anti-TPBG antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-TPBG antibody.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • An "individual" according to any of the above embodiments is preferably a human.
  • the invention provides for the use of an anti-TPBG antibody in the manufacture or preparation of a medicament.
  • the medicament is for treatment of cancer.
  • the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • An "individual" according to any of the above embodiments may be a human.
  • the invention provides a method for treating cancer.
  • the method comprises administering to an individual having cancer an effective amount of an anti-TPBG antibody.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
  • An "individual" according to any of the above embodiments may be a human.
  • the invention provides pharmaceutical formulations comprising any of the anti-TPBG antibodies provided herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical formulation comprises any of the anti-TPBG antibodies provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprises any of the anti-TPBG antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
  • Antibodies of the invention can be used either alone or in combination with other agents in a therapy.
  • an antibody of the invention may be co-administered with at least one additional therapeutic agent.
  • an additional therapeutic agent is a chemotherapeutic agent.
  • chemotherapeutic agents include, but are not limited to, anti-neoplastic agents including alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal(TM) (temozolamide), ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcita
  • the chemotherapeutic agent is selected from the group consisting of taxanes (like e.g. paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine.
  • the chemotherapeutic agent is selected from the group consisting of taxanes (like e.g.
  • the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin, irinotecan, or oxaliplatin.
  • the chemotherapeutic agent is 5-fluorouracil, leucovorin and irinotecan (FOLFIRI).
  • the chemotherapeutic agent is 5-fluorouracil, and oxaliplatin (FOLFOX).
  • therapeutic appliacations of antibodies of the invention with additional chemotherapeutic agents include, for instance,
  • the additional chemotherapeutic agent is selected from the group of taxanes (docetaxel or paclitaxel or a modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine and/or bevacizumab for the treatment of breast cancer.
  • the antibodies of the invention are administered in absence of an additional chemotherapeutic agent.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • Antibodies of the invention can also be used in combination with radiation therapy.
  • An antibody of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an antibody of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.5mg/kg - 10 mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks ( e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody).
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention or a recombinant fusion protein of the invention in place of or in addition to an anti-TPBG antibody.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bac
  • any of the above articles of manufacture may include an immunoconjugate of the invention or a recombinant fusion protein of the invention in place of or in addition to an anti-TPBG antibody.
  • DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
  • Desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis.
  • the gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids.
  • the plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the sub-cloned gene fragments was confirmed by DNA sequencing.
  • Cynomolgus monkey was previously used to evaluate TPBG-specific antibodies or conjugates thereof for their tolerability ( Sapra et al. (2013) Mol Canc Ther 12: 38-47 ). 14 different tissues of cynomolgus monkey and minipig were prepared and the mRNA expression profile evaluated. Transcription profiling by sequencing of tissue RNAs coming from Roche cynomolgus monkey and minipig RNAseq-projects was evaluated on Illumina NextSeq500 as indexed, 75bp, paired-end run with an average depth of 30 million reads pairs. Gene expression signals are indicated as unique reads per kilobase exon model per million reads (rpkm) in log scale indicating median expression levels and standard deviation.
  • minipig is a suitable toxicology species to evaluate novel minipig crossreactive anti-TPBG antibodies and Fab-PE constructs ( Figure 1 ).
  • the cDNA of either full length TPBG, thus comprising extracellular domain, transmembrane domain and intracellular domain or fragments thereof was cloned in eukaryotic expression vectors. Cloning was performed by standard restriction digest. For plasmids containing a C-terminal PreScission site followed by a HIS 6 -Avi epitope tag or a C-terminal PreScission site followed by a huIgG 1 -Fc-HIS 6 epitope tag, site-directed cloning was performed with BamHI and NotI restriction enzymes. Plasmids were sequence verified and amplified according to standard molecular biology techniques.
  • Plasmids containing the extracellular domain of TPBG or fragments thereof were transiently transfected in HEK293F (Life Technologies) cells. Briefly, cells were cultured in FreeStyle 293-F (Life Technologies) expression medium. The day before transfection cells were seeded at a density of 1x10 6 /ml culture medium. The following day 1 ⁇ g of plasmid DNA was used per ml culture medium. 293Free (Life Technologies) transfection reagent was used for transient transfection. Transfected cells were incubated at 37°C and 8% CO 2 for 7d while shaking at 125-130 rpm in Erlenmeyer flasks. Supernatant was harvested and either frozen at - 80°C or directly processed for protein purification.
  • TPBG containing a huIgG 1 -Fc-HIS 6 epitope tag human TPBG: SEQ ID NO: 28; minipig TPGB: SEQ ID NO: 30; cynomolgus TPBG: SEQ ID NO: 32; murine TPBG: SEQ ID NO: 34, rat TPBG: SEQ ID NO: 36; the listed sequences all include signal peptides
  • TPBG containing a huIgG 1 -Fc-HIS 6 epitope tag
  • Recombinant TPBG containing a HIS 6 -Avi epitope tag (human TPBG: SEQ ID NO: 27; minipig TPGB: SEQ ID NO: 29; cynomolgus TPBG: SEQ ID NO: 31; murine TPBG: SEQ ID NO: 33, rat TPBG: SEQ ID NO: 35 , the listed sequences all include signal peptides) was purified from cell culture supernatant using a HisTrap HP (GE Healthcare) column. Elution occurred with an imidazole step gradient. Affinity purified protein was subsequently loaded on a HiLoad 16/60 Superdex 200 prep grade (GE Healthcare) size exclusion column. The product peak was collected and volume adjusted by ultracentrifugation with Amicon Ultra Filters (Merck Millipore). The final storage buffer consisted of 20 mM Histidine, 140 mM NaCl, pH 5.5.
  • One set of 3 rabbits was immunized with 400 ⁇ g of recombinant human TPBG extracellular domain (ECD) coupled to huFc, emulsified with complete Freund's adjuvant, at day 0 by intradermal application, and with 200 ⁇ g each of TPBG-huFc, emulsified with complete Freund's adjuvant, at days 7, 14, 28, 56 and 84, by alternating intramuscular and subcutaneous applications.
  • Blood (10% of estimated total blood volume) was taken at days 20, 34, 62 and 90. Serum was prepared, which was used for titer determination by ELISA (see below), and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process.
  • Another set of 3 rabbits was immunized genetically, using a plasmid expression vector coding for full-length human TPBG, by intradermal application of 400 ⁇ g vector DNA, followed by electroporation (5 square pulses of 750 V/cm, duration 10 ms, interval 1 s). Rabbits received 7 consecutive immunizations at days 0, 14, 28, 49, 70, 98 and 126. Blood (10% of estimated total blood volume) was taken at days 35, 77, 105 and 133. Serum was prepared, which was used for titer determination by ELISA (see below), and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process.
  • Human recombinant soluble TPBG extracellular domain was immobilized on a 96-well NUNC Maxisorp plate at 2 ⁇ g/ml, 100 ⁇ l/well, in PBS, followed by: blocking of the plate with 2% Crotein C in PBS, 200 ⁇ l/well; application of serial dilutions of antisera, in duplicates, in 0.5% Crotein C in PBS, 100 ⁇ l/well; detection with either (1) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch/Dianova), or (2) HRP-conjugated rabbit anti-human IgG antibody (Pierce/Thermo Scientific; 1/5000), or (3) biotinylated goat anti-human kappa antibody (Southern Biotech/Biozol; 1/5000) and streptavidin-HRP; each diluted in 0.5% Crotein C in PBS, 100 ⁇ l/well.
  • EDTA-containing whole blood was diluted twofold with 1x PBS (PAA, Pasching, Austria) before density centrifugation using lympholyte mammal (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the specifications of the manufacturer.
  • PBMCs were washed twice with 1x PBS.
  • Sterile streptavidin coated 6-well plates (Microcoat, Bernried, Germany) were coated with 2 ⁇ g/ml of the biotinylated extracellular domain of human TPBG protein in PBS for 3 h at room temperature. Prior to the panning step these 6-well plates were washed three times with sterile PBS.
  • the PBMCs were seeded on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes through unspecific adhesion. Each well was filled at maximum with 4 ml medium and up to 6 x 10e6 PBMCs from the immunized rabbit and were allowed to bind for 1 h at 37°C and 5% CO 2 .
  • the cells in the supernatant peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.
  • 6-well plates coated with TPBG protein were seeded with up to 6 x 10e6 PBLs per 4 ml medium and allowed to bind for 1 h at 37 °C and 5% CO 2 to enrich TPBG-specific B cells.
  • Non-adherent cells were removed by carefully washing the wells 1-2 times with 1x PBS. The remaining sticky cells were detached by trypsin for 10 min at 37 °C and 5% CO 2 . Trypsination was stopped with EL-4 B5 medium (EL-4 B5: RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 2 mM Glutamin, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0,05 mM b-mercaptoethanole (Gibco, Paisley, Scotland).
  • EL-4 B5 RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 2 mM Glutamin,
  • the cells were kept on ice until the immune fluorescence staining.
  • Anti-IgG FITC AbD Serotec, Dusseldorf, Germany
  • For surface staining cells from the depletion and enrichment step were incubated with anti-IgG FITC antibody in PBS and incubated for 45 min in the dark at 4°C. After staining the PBLs were washed two times with ice cold PBS. Finally the PBLs were resuspended in ice cold PBS and immediately subjected to the FACS analyses.
  • Propidium iodide at a concentration of 5 ⁇ g/ml was added prior to FACS analyses to discriminate between dead and live cells.
  • a Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences, USA) was used for single cell sort.
  • the cultivation of the rabbit B cells was done by a method described by Seeber S. et al., PLoS One. 2014 Feb 4;9(2):e86184 .
  • single sorted rabbit B cells were incubated in 96-well plates with 200 ⁇ l/well EL-4 B5 medium containing Pansorbin cells (1:100000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (MicroCoat, Bernried, Germany) and gamma-irradiated murine EL-4 B5 thymoma cells (2.5 ⁇ 10e4 cells/well) for 7 days at 37 °C in the incubator.
  • the supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and were frozen at - 80 °C in 100 ⁇ l RLT buffer (Qiagen, Hilden, Germany).
  • the PCR conditions for the RbVH+RbVL were as follows: Hot start at 94°C for 5 min; 35 cycles of 20s at 94°C, 20s at 70°C, 45s at 68 °C, and a final extension at 68°C for 7 min.
  • the PCR conditions for the HuVL were as follows: Hot start at 94°C for 5 min; 40 cycles of 20s at 94°C, 20s at 52°C, 45s at 68 °C, and a final extension at 68°C for 7 min.
  • Primer sequences rbHC.up AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC rbHCf.do CCATTGGTGAGGGTGCCCGAG BcPCR_FHLC_leader.fw ATGGACATGAGGGTCCCCGC BcPCR_huCkappa.rev GATTTCAACTGCTCATCAGATGGC
  • anti-TPBG samples were added in a 1:2 dilution series starting at 2 ⁇ g/ml and incubated 1h at RT.
  • 25 ⁇ l/well goat anti c-myc HRP (Bethyl, # A190-104P) or goat anti hu kappa HRP (Millipore, # AP502P) was added in a 1:7000 or 1:4000 dilution, respectively and incubated at RT for 1 h on a shaker.
  • TMB substrate (Calbiochem, #CL07) was added and incubated 2 min for human and minipig TPBG, 3 min for cyno TPBG and 4 min for mouse and rat TPBG. Measurement took place at 370/492 nm on a Safire2 reader (Tecan).
  • the human breast cancer tumor cell line MFC7 endogenously expressing TPBG was seeded at a concentration of 21000 cells/well in 384-well cellcoat Poly-D-Lysine plates (Greiner, #781940). Cells were allowed to attach over night at 37°C. After removing the supernatant, 25 ⁇ l/well of supernatant containing anti-TPBG antibodies were added in a 1:2 dilution series starting at 5 ⁇ g/ml and incubated 1h at 4°C.
  • Table 1 Binding of anti-TPBG Fab fragments to TPBG of different species EC50 [ng/ml] recombinant TPBG MCF7 sample: huTPBG muTPBG rtTPBG cyTPBG mpTPBG huTPBG 051 18.1 - - >2000 175.0 57.5 091 27.7 - - 1229.8 61.8 14.0 097 15.2 - - - 43.6 451.3
  • Fab fragments of 051, 091, and 097 were found to be crossreactive between human and minipig TPBG. Cell binding of human TPBG expressed on cells of a human breast cancer cell line was confirmed.
  • Nunc maxisorp plates (Nunc, 464718) were coated with 25 ⁇ l/well Fc-TPBG at a concentration of 1 ⁇ g/ml for rat, minipig, mouse, cyno and at a concentration of 500 ng/ml for human TPBG. Plates were incubated at 4°C overnight. After washing (3x 90 ⁇ l/well; PBS-T buffer) plates were blocked with 90 ⁇ l blocking buffer (PBS with 2 % BSA and 0.05 % Tween-20) for 1h at room temperature. After washing (3x90 ⁇ l/well with PBST-buffer) anti-TPBG antibodies were added in a 1:2 dilution series starting at 1 ⁇ g/ml and incubated 1h at RT.
  • 384-well TRSA-SA Maxisorp (MicroCoat) plates were coated with 25 ⁇ l/well biotinylated human TPBG-AviHis at a concentration of 200 ng/ml followed by an incubation at 4°C overnight. After washing 3 times with 90 ⁇ l/well PBS-T, antibodies A1, A2, A3 were added at a concentration of 4 ⁇ g/ml followed by an incubation for 1h at room temperature. Wells were washed 3 times with 90 ⁇ l/well PBS-T. Anti-TPBG antibodies were added at a concentration of 0.1 ⁇ g/ml and incubated for 1 h at room temperature on a shaker.
  • PCR-products coding for VH or VL were cloned as cDNA into expression vectors by the overhang cloning method ( RS Haun et al., Biotechniques (1992) 13, 515-518 ; MZ Li et al., Nature Methods (2007) 4, 251-256 ).
  • the expression vectors contained an expression cassette consisting of a 5' CMV promoter including intron A, and a 3' BGH poly adenylation sequence.
  • the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E.coli.
  • Three variants of the basic plasmid were used: one plasmid containing the rabbit IgG constant region designed to accept the VH regions while two additional plasmids containing rabbit or human kappa LC constant region to accept the VL regions.
  • Linearized expression plasmids coding for the kappa or gamma constant region and VL /VH inserts were amplified by PCR using overlapping primers.
  • plasmid and insert were combined and incubated with recA which induced site specific recombination.
  • the recombined plasmids were transformed into E.coli.
  • the isolated HC and LC plasmids were transiently co-transfected into HEK293 cells and the supernatants were harvested after 1 week.
  • the PCR conditions for the VH and VL chain amplification were as follows: Hot start at 94°C for 5 min; 35 cycles of 20s at 94°C, 20s at 68°C, 45s at 68 °C, and a final extension at 68°C for 7 min.
  • PCR-products coding for VH or VL were cloned as cDNA into expression vectors by the overhang cloning method ( RS Haun et al., Biotechniques (1992) 13, 515-518 ; MZ Li et al., Nature Methods (2007) 4, 251-256 ).
  • the expression vectors contained an expression cassette consisting of a 5' CMV promoter including intron A, and a 3' BGH poly adenylation sequence.
  • the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E.coli.
  • Two variants of the basic plasmid were used: one plasmid containing the human IgG constant region designed to accept the new amplified VH chain and a second plasmid containing the human kappa LC constant region to accept the VL chain.
  • Linearized expression plasmids coding for the kappa or gamma constant region and VL /VH inserts were amplified by PCR using overlapping primers.
  • plasmid and insert were combined and incubated with recA which induced site specific recombination.
  • the recombined plasmids were transformed into E.coli.
  • An immunoconjugate of an anti-TPBG antibody Fab fragment and a truncated variant of exotoxin A chain of P. aeruginosa was generated via enzymatic coupling with sortase.
  • Fab fragments transiently expressed in HEK293-F (with LPETG-HIS6 motif at the C-terminus) and GGG-PE24 LR8M (purified from E. coli extract as published in the literature) were concentrated to 7 - 8 mg/ml in 20 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5. Both proteins were combined in a 1:1 molar ratio. Coupling reaction was started by adding HIS-tagged Sortase (0.8 molar equivalent). After incubation for 1 h at 37°C the reaction mixture was chromatographed on a Ni HiTrap column (GE Healthcare) to remove uncoupled Fab and Sortase.
  • Coupled Fab-PE24 eluted in the flow through and was further purified on a Superdex200 gel filtration column in 20 mM His, 140 mM NaCl, pH 6.0. The final product was concentrated to 1 mg/ml and frozen in aliquots at -80°C.
  • An immunoconjugate of an anti-TPBG antibody Fab fragment and a truncated variant of exotoxin A chain of P. aeruginosa was generated via expression of the immunoconjugate as recombinant fusion protein in E.coli cells.
  • the toxin was fused to the C-terminus of the heavy chain of the anti-TPBG antibody.
  • variable light chain constructs LC
  • variable heavy chain constructs HC-PE
  • LC variable light chain constructs
  • HC-PE variable heavy chain constructs
  • the E.coli K12 strain CSPZ-25 (thi-1, ⁇ ompT, ⁇ pyrF, ⁇ lon, ⁇ galE) was transformed by electroporation with the relevant expression plasmids.
  • the transformed E.coli cells were first grown at 37 °C on agar plates. For each transformation a colony picked from this plate was transferred to a 3 mL roller culture and grown at 37°C to an optical density of 1-2 (measured at 578 nm). Then 1000 ⁇ l culture were mixed with 1000 ⁇ l sterile 86%-glycerol and immediately frozen at -80°C for long time storage. The correct product expression of these clones was verified in small scale shake flask experiments and analyzed with SDS-Page.
  • IBP Inclusion body preparations of the 10 L fermentations were routinely started directly after the harvest of the bacteria with the re-suspension of the cell pellet in buffer containing 12.1 g/l Tris, 0.246 g/l MgSO4*7H2O and 12ml/l 25%-HCl.
  • the buffer volume was calculated in dependence of the dry matter content of the biomass. Lysozyme (100 kU/mg) and a small amount of BenzonaseTM were added. Then the suspension was homogenized at 900 bar (APV Rannie 5, 1 pass) to disrupt the bacteria cells followed by further addition of Benzonase and an incubation for 30 min at 30-37°C.
  • the first wash buffer 60g/l Brij, 87.6g/l NaCl, 22.5g/l EDTA, 6ml/l 10N NaOH
  • the second wash buffer (12.1g/l Tris, 7.4g/l EDTA, 11ml/1 25%-HCl) and incubated for 20 min.
  • a further separation step yielded the inclusion bodies which are stored frozen at -20°C or immediately transferred to the DSP department.
  • Inclusion bodies of HC-PE and LC were solubilized separately in 6.7 M Guanidinium-Hydrochloride, 100 mM Tris/HCl, 5 mM acetate, 1mM EDTA pH 8.0 + 100 mM Dithiothreitol (DTT) 2h at room temperature. Solubilized material was adjusted to pH 5 and microfiltrated (6.7 M Guanidinium-HCl, 100mM Tris, 1 mM EDTA, 5mM acetate, 100mM DTT pH 5.0) using a 1000 kDa membrane.
  • the renaturation solution was kept at 2 - 10°C overnight.
  • the renaturate was diafiltrated against 20 mM Tris/HCl, 40 mM NaCl pH7.4 (pH 8.0 for TPBG-0199) and pumped onto an anion exchange column (AIEX) equilibrated in 20 mM Tris/HCl, 40mM NaCl pH 7.4 (pH 8.0 for TPBG-0199). After washing the column with equilibration buffer the protein was eluted with a gradient up to 20 mM Tris/HCl, 320 mM NaCl, pH 7.4 (pH 8.0 for TPBG-0199).
  • Peak fractions containing Fab-PE were pooled, concentrated and applied onto a preparative Size Exclusion Column (SEC) in 20 mM Tris, 150 mM NaCl, pH 7.4 to remove aggregates, fragments and E. coli proteins.
  • SEC Size Exclusion Column
  • the final protein pool was adjusted to the required protein concentration and analyzed via Bioanalyzer (Agilent Technologies), analytical SEC and UV 280 , identity was confirmed by mass spectrometry
  • ESI Electrospray ionization
  • MS parameter settings were as follows: Transfer: Funnel RF, 400 Vpp; ISCID Energy, 0 or 85 eV; Multipole RF, 400 Vpp; Quadrupole: Ion Energy, 3.0 eV; Low Mass, 850 m/z; Source: Dry Gas, 8 L/min; Dry Gas Temperature, 160°C; Collision Cell: Collision Energy, 8 eV; Collision RF: 3800 Vpp; Ion Cooler: Ion Cooler RF, 800 Vpp; Transfer Time: 140 ⁇ s; Pre Puls Storage, 20 ⁇ s; scan ranges m/z 600 to 2000 and m/z 1000 to 4000.
  • the MassAnalyzer software (developed in-house) was used for data evaluation.
  • CHO-K1 cells were stably transfected with full length TPBG from different species, namely human, or minipig, to evaluate cellular binding properties and potency of novel TPBG-specific antibodies.
  • CHO-K1 cells ATCC
  • DMEM/F12 medium no phenol red
  • fetal calf serum 10% fetal calf serum
  • 2 mM L-Glutamine 10% fetal calf serum
  • 80-90% confluency lipid/DNA complexes were prepared with TPBG expression vectors using Roche's XtremeGeneHP transfection reagent.
  • lipid/DNA complexes were added to 2x10 5 CHO-K1 cells in suspension at a total volume of 2 ml of selection medium (DMEM/F12, 10% fetal calf serum, 400 ⁇ g/ml G418). Cells were cultivated for 14 days under selection pressure and then stained with in-house TPBG antibodies. Briefly, 2x10 5 cells were stained with 2 ⁇ g/ml primary anti-TPBG antibody for 30 min on ice, washed and counterstained with PE-labelled anti-human kappa antibody (1:50 in PBS containing 2% fetal calf serum, 30min on ice).
  • Single cell sorting of high or medium to low expressing clones was performed on a FacsARIA (BD) and single cells were deposited into 96-well flat bottom cell culture plates. After 12 days a variety of clones for each species (human, cynomolgus, minipig TPBG) were transferred to 24-well plates and cultivated for three more days. Clones were stained with above mentioned internal reference TPBG antibodies as formerly described and analyzed on a FacsCanto (BD). Selected clones were cultivated for one more week and stored in FCS containing 7.5% DMSO in liqid nitrogen. For stability testing, cells were further cultivated for two more weeks, then stained and analyzed on a FacsCanto (BD) resulting in the final selection of TPBG-expressing CHO cells that were stable over the observed time period.
  • FacsARIA FacsARIA
  • a SW620 clone (clone 4), stably expressing high level of TPBG, was chosen as cellular model.
  • Cells growing in the logarithmic growth phase were detached using Accutase (Sigma) and a cell suspension was prepared in a PBS buffer (bufferA) containing 1 % fetal calf serum and pH adjusted to pH 7.2, 6.0, and 5.5. Respective Fabs were added to cell suspension and binding occurred for 30 min on ice. Cells were subsequently washed twice with buffer of the same pH (buffer A).
  • Table 8 Comparison of anti-TPBG Fab binding to cell surface TPBG at different pH values Fab SW620 cl.4 Fold change pH 7.2 vs pH 5.5 replicate 1 replicate 2 H8 4.2 6.8 051 1.1 1.1 091 0.9 1.0 097 1.2 1.2
  • the GeoMean fluorescence signal of each Fab sample was corrected by the mean fluorescence intensity (MFI) of the corresponding isotype control.
  • MFI mean fluorescence intensity
  • the MFI for each time point was set in relation to time point zero and calculated as percentage value.
  • the percentage of internalization was calculated by subtracting the percentage of detected antibody from 100%.
  • TPBG-specific Fabs as expressed in Example 11 was assessed using stably transfected CHO cells (Example 16) and a flow cytometry based assay.
  • Stable transfectants expressing cell surface levels of TPBG in a similar range were used for the flow cytometry based analysis of cellular binding.
  • Dilution series of antibodies were prepared spanning a concentration range from 0.01 - 20 ⁇ g/ml.
  • cell suspensions were prepared in ice cold PBS (Gibco) containing 2 % fetal calf serum (PAN Biotech).
  • 2.5x10e5 cells were dispensed per well in a v-shaped 96well-MTP.
  • Antibody dilution series prepared in the same buffer were added as twofold concentrated solution. Samples were incubated on ice for 45 min upon which they were washed twice with 150 ⁇ L ice cold PBS containing 2% fetal calf serum.
  • Antibodies 051, 091 and 097 of the invention bound to cell surface expressed TPBG to a similar degree, while prior art antibody H8 did not bind to cell surface expressed TPBG in sufficient extent ( Figure 4A ) .
  • TPBG-specific antibodies to either human (hu) or minipig (pg) TPBG was evaluated using CHO-K1 cells stably transfected with the respective full length antigen (Example 16). Stable transfectants expressing cell surface levels of TPBG in a similar range were used for flow cytometry based analysis of cellular binding. Dilution series of Fab-PEs (anti-TPBG Fab-PE fusion proteins as expressed in Examples 13 and 14) were prepared spanning a concentration range from 400 - 0.002 nM. In brief, cell suspensions were prepared in ice cold PBS (Gibco) containing 2 % fetal calf serum (PAN Biotech).
  • 2.5x10e5 cells were dispensed per well in a v-shaped 96well-MTP.
  • Fab-PE dilution series prepared in the same buffer were added as twofold concentrated solution. Samples were incubated on ice for 45 min upon which they were washed twice with 150 ⁇ L ice cold PBS containing 2% fetal calf serum. In between, cells were spun down at 300 g for 5 min at 4°C. Cells were resuspended in 50 ⁇ L ice-cold buffer containing 10 ⁇ g / mL anti-kappa light chain RE-PE (phycoerythrin) labeled secondary antibody (Life Technologies). The suspension was incubated for 45 min on ice.
  • RE-PE phytoerythrin
  • Fab-PE constructs Fabs coupled to PE fragment via sortase coupling as provided in Example 12
  • pg minipig
  • CHO-K1 cells were transiently transfected with TPBG and luciferase containing plasmids in a ratio of 2:1 in suspension using Lipofectamine 3000 at a ratio of 3:1 and 3 ⁇ g total DNA per 1x10e6 cells. 30.000 cells per well were then seeded in a 98well-MTP in 100 ⁇ L medium (Gibco) containing 10% fetal calf serum (PAN Biotech). Cells were allowed to attach for 21 h prior addition of compounds. Dilution series of Fab-PEs were prepared in medium spanning a concentration range from 15 to 0.0003 nM. Individual data points were measured as triplicates.
  • luciferase reporter activity as a surrogate marker for cellular protein synthesis capacity was quantified using Steady Glo (Promega). Luminescence was recorded in a microplate reader (Tecan). Data analysis was performed with Excel (Microsoft) and XLfit 5.3.1 (IDBS) add-in. Experiments were performed as biological replicates.
  • TPBG-specific Fabs were co-incubated in a 1:3 molar ratio for 30 min prior addition to cells with a second Fab binding to the kappa-light chain of the TPBG-specific Fab.
  • the kappa binding Fab carried in addition a pseudomonas exotoxin (PE) moiety.
  • PE pseudomonas exotoxin
  • the so formed complex was added to CHO-K1 cells stably expressing either human or minipig TPBG.
  • CHO-K1 cells were stably transfected with full length TPBG from the respective species (Example 16). Single clones were selected and expanded. Clones expressing cell surface TPBG in a similar range were selected for subsequent proliferation assays. While the clones were maintained under selection pressure, the actual proliferation assays were performed in medium without selection pressure.
  • Fabs derived vom antibodies A1, A2, and A3 do not display significant potency on CHO-K1 stably transfected with minipig TPBG. Also, the Fab fragment derived from antibody H8 displays a significantly lower potency in this assay and has an about 100x lower potency on cells expressing minipig than on cell expressing human TPBG ( Figure 6A-D ).
  • Table 11 Cytotoxic activity of anti-TPBG Fab-PE sandwich constructs on human and minipig TPBG expressing CHO cells (EC50 in [nM]) Fab + anti-kappa-Fab-PE CHO-K1 / human TPBG CHO-K1 / minipig TPBG replicate 1 replicate 2 replicate 1 replicate 2 Unspecific Fab - - - - H8 0.41 0.52 47.97 45.86 A1 3.63 4.12 - - A2 9.81 11.33 - - A3 0.26 0.47 - - 051 0.28 0.38 6.18 5.04 091 0.23 0.24 1.05 1.20 097 0.30 0.33 2.08 2.15
  • a surrogate assay to evaluate the potency of Fab-PE constructs (anti-TPBG Fab-PE fusion proteins as expressed in Examples 13 and 14) on cells expressing human (hu) or minipig (pg) TPBG was established.
  • CHO-K1 cells were stably transfected with full length TPBG from the respective species. Single clones were selected and expanded. Clones expressing cell surface TPBG in a similar range were selected for subsequent proliferation assays. While the clones were maintained under selection pressure, the actual proliferation assays were performed in medium without selection pressure.
  • All immunoconjugates comprising antibodies of the invention (i.e. antibodies 051, 091, and 097) displayed potent activity on cells expressing either human or minipig TPBG.
  • An unspecific Fab-PE which does not bind to transfected CHO-K1 cells has no activity in the measured concentration range ( Figures 7 A) .
  • Table 12 Cytotoxic activity of anti-TPBG Fab-PE fusion proteins on CHO cells stably expressing either human or minipig TPBG (EC50 in [nM]) Fab-PE comprising antibody stable CHO-K1 transfectants X-fold lower potency pgTPBG versus huTPBG huTPBG pgTPBG Unspecific Fab - - H8 0.07 5.98 83.9 051 0.03 1.37 51.7 091 0.05 0.15 3.3 097 0.04 0.14 3.4
  • Binding of anti-TPBG Fab-PE fusion proteins as provided in Example 13 and 14, as well as binding of Fabs coupled to PE fragment via sortase coupling as provided in Example 12 to human tumor cells was studied using the breast cancer tumor cell line MCF7. Cells growing in the logarithmic growth pase were detached using Accutase (Sigma). In brief, cell suspensions were prepared in ice cold PBS (Gibco) containing 2 % fetal calf serum (PAN Biotech).
  • 2.5x10e5 cells were dispensed per well in a v-shaped 96well-MTP.
  • Fab-PE dilution series ranging from 133 - 0.001 nM prepared in the same buffer were added as twofold concentrated solution. Samples were incubated on ice for 45 min upon which they were washed twice with 150 ⁇ L ice cold PBS containing 2% fetal calf serum. In between, cells were spun down at 300 g for 5 min at 4°C. Cells were resuspended in 50 ⁇ L ice-cold buffer containing 10 ⁇ g / mL anti-kappa light chain RE-PE (phycoerythrin) labeled secondary antibody (Life Technologies).
  • RE-PE phytoerythrin
  • the suspension was incubated for 45 min on ice.
  • Cells were washed twice as described and resuspended in 50 ⁇ L ice-cold buffer containing 1 ⁇ g/mL Hoechst 33258. After 10 min cells were washed once more and finally resuspended in 200 ⁇ L ice-cold fixation buffer (BD CellFix). Samples were measured on a FACSCanto II (BD). Data processing was done using the FlowJo Software. Data analysis was performed with Excel (Microsoft) and XLfit 5.3.1 (IDBS) add-in.
  • All anti-TPBG Fab-PE fusion proteins display low nM or sub-nM EC50 values in their binding to the human breast tumor cell line MCF7 ( Figure 8 ).
  • Table 13 Cellular binding of anti-TPBG Fab-PE fusion proteins on human TPBG expressing MCF7 cells (EC50 in [nM]) Fab comprised in immunoconjugate type of immunoconjugate EC50 [nM] H8 sortase-coupled 1.56 051 sortase-coupled 0.67 refolded 1.25 091 sortase-coupled 0.61 refolded 0.89 097 sortase-coupled 0.63 refolded 0.64
  • TPBG-specific Fabs were co-incubated in a 1:3 molar ratio for 30 min prior addition to cells with a second Fab binding to the kappa-light chain of the TPBG-specific Fab.
  • the kappa binding Fab carried in addition a pseudomonas exotoxin (PE) moiety.
  • PE pseudomonas exotoxin
  • TPBG-specific Fabs were co-incubated in a 1:3 molar ratio for 30 min prior addition to cells with a second Fab binding to the kappa-light chain of the TPBG-specific Fab.
  • the kappa binding Fab carried in addition a pseudomonas exotoxin (PE) moiety.
  • PE pseudomonas exotoxin
  • Fab-PE constructs Fabs coupled to PE fragment via sortase coupling as provided in Example 12
  • the breast cancer cell line MCF7 was selected. 20.000 cells were seeded per well in a 98well-MTP in 100 ⁇ L medium (Gibco) containing 10% fetal calf serum (PAN Biotech). Cells were allowed to attach for 24 h prior addition of compounds. Dilution series of sortase-coupled Fab-PEs were prepared in medium spanning a concentration range from 133 to 0.002 nM. Individual data points were measured as triplicates. 72 h after compound addition CellTiter-Glo (Promega) assay was performed. Luminescence was recorded in a microplate reader (Tecan). Data analysis was performed with Excel (Microsoft) and XLfit 5.3.1 (IDBS) add-in. Experiments were performed as biological replicates.
  • Table 16 Cytotoxic activity of sortase coupled anti-TPBG Fab-PE constructs on human TPBG expressing MCF7 cells (EC50 in [nM]) Fab-PE comprising antibody MCF7 X-fold higher potency compared to TPBG(H8)PE Unspecific Fab - H8 0.059 - 051 0.036 1.7 091 0.033 1.8 097 0.054 1.1
  • TPBG-specific Fabs expressed as described in Example 11 were co-incubated in a 1:3 molar ratio for 30 min prior addition to cells with a second Fab binding to the kappa-light chain of the TPBG-specific Fab.
  • the kappa binding Fab carried in addition a pseudomonas exotoxin (PE) moiety.
  • PE pseudomonas exotoxin
  • the so formed complex was added to human H1975 lung cancer cells.
  • 10.000 cells were seeded per well in a 98well-MTP in 100 ⁇ L medium (Gibco) containing 10% fetal calf serum (PAN Biotech) and penicillin/streptomycin (Gibco). Cells were allowed to attach for 24 h prior addition of compounds.
  • Dilution series were prepared in medium spanning a concentration range from 133 to 0.002 nM as calculated for the respective TPBG-specific Fab. Individual data points were measured as triplicates. 72 h after compound addition CellTiter-Glo (Promega) assay was performed. Luminescence was recorded in a microplate reader (Tecan). Data analysis was performed with Excel (Microsoft) and XLfit 5.3.1 (IDBS) add-in. Experiments were performed as biological replicates.
  • Sandwich constructs comprising Fab fragments of prior art antibodies A1, A2 and A3 displayed no (for A1 and A2) or significantly lower cytotoxicity than the other tested constructs ( Figure 12 ).
  • Table 17 Cytotoxic activity mediated by anti-TPBG Fab-PE sandwich constructs on human TPBG expressing H1975 cells (EC50 in [nM]) Fab of antibody H1975 replicate 1 replicate 2 replicate 3 H8 0.698 0.451 0.656 051 0.522 0.397 0.527 091 0.166 0.129 0.182 097 0.826 0.656 0.706 A1 - - - A2 - - - A3 3.444 2.765 > 3
  • TPBG tumor cell line H1975
  • the number of cell surface TPBG molecules was approximately 30.000 / cell as determined by flow cytometry based receptor quantitation. 10.000 cells were seeded per well in a 98well-MTP in 100 ⁇ L medium (Gibco) containing 10% fetal calf serum (PAN Biotech). Cells were allowed to attach for 24 h prior addition of compounds. Dilution series of Fab-PEs were prepared in medium spanning a concentration range from 3 to 0.00005 nM. Individual data points were measured as triplicates.
  • Table 18 Cytotoxic activity mediated by anti-TPBG Fab-PE fusion proteins on human TPBG expressing H1975 cells (EC50 in [nM]) Fab of antibody H1975 X-fold higher potency compared to Fab-PE fusion protein of H8 replicate 1 replicate 2 Unspecific Fab - - H8 0.019 0.013 - 051 0.016 0.014 1.2 / 1.1 091 0.005 0.003 3.8 / 3.7 097 0.013 0.013 1.4 / 1.1
  • Example 13 and 14 In a different experimental setup the exposure time of of anti-TPBG Fab-PE fusion proteins as expressed in Example 13 and 14 to tumor cells was varied.
  • the non-small cell lung cancer cell line H1975 was used to evaluate, if shorter incubation time with Fab-PE constructs has an impact on potency in a proliferation assay.
  • Table 19 Time course proliferation experiment of human TPBG expressing H1975 cells contacted with anti-TPBG Fab-PE fusion proteins (EC50 in [nM]) Fab comprised in immunoconjugate H1975 continuous 60 min 30 min 10 min H8 0.013 0.180 0.201 0.229 051 0.014 0.180 0.204 0.241 091 0.003 0.043 0.062 0.057 097 0.013 0.203 0.189 0.211
  • Table 20 Time course proliferation experiment of human TPBG expressing H1975 cells contacted with anti-TPBG Fab-PE fusion proteins (X-fold hihger potency than anti-TPBG Fab-PE fusion protein of prior art antibody H8) Fab comprised in immunoconjugate H1975 continuous 60 min 30 min 10 min H8 1.0 1.0 1.0 1.0 051 1.1 1.0 1.0 1.1 091 3.7 4.2 3.3 4.0 097 1.1 1.1 0.9 0.9 0.9
  • NCI-H1975 displays cell surface TPBG as was independently confirmed.
  • NCI-H1975 were cultured in RPMI high glucose medium with 1.0 mM sodium pyruvate supplemented with 10 % fetal calf serum, 2.0 mM L-glutamine, and 10 mM HEPES at 37 °C in a water-saturated atmosphere at 5 % CO 2 . Culture passages were performed with trypsin / EDTA (Life Technologies) splitting every third day. Nude mice were purchased from breeder (e.g. Charles River, Sulzfeld, Germany) and maintained under specific-pathogen-free condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government.
  • Fab-PE fusion proteins were administered as single agent at 1 mg/kg i.v. once daily every second day for several times. The corresponding vehicle was administered on the same days.
  • Fab-PE proteins were provided as stock solution from Roche, Penzberg, Germany. Buffer included histidine and injection solution was diluted appropriately in buffer from stock prior injections.
  • NCI-H1975 NSCLC xenograft bearing mice were treated with the Fab-PE fusion proteins from study day 17 to 44 at dose of 1.0 mg/kg for a total of 8 times (days 17, 19, 21, 24, 26, 28, 42 and 44). Each treatment group consisted of 10 animals.
  • treatments with the Fab-PE fusion proteins comprising antibodies of the invention showed significant anti-tumor efficacy with strong tumor regression of s.c. NCI-H1975 xenografts. Notably, a majority of complete tumor remissions were achieved.
  • NCI-N87 were cultured in RPMI high glucose medium with 1.0 mM sodium pyruvate supplemented with 10 % fetal calf serum, 2.0 mM L-glutamine, and 10 mM HEPES at 37 °C in a water-saturated atmosphere at 5 % CO 2 . Culture passages were performed with trypsin / EDTA (Life Technologies) splitting every third day. Nude mice were purchased from breeder (e.g. Charles River, Sulzfeld, Germany) and maintained under specific-pathogen-free condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). Experimental study protocol was reviewed and approved by local government.
  • Fab-PE fusion proteins were administered as single agent at 1 mg/kg i.v. once daily every second day for several times. The corresponding vehicle was administered on the same days.
  • Fab-PE fusion proteins were provided as stock solution from Roche, Penzberg, Germany. Buffer included histidine and injection solution was diluted appropriately in buffer from stock prior injections.
  • NCI-N87 gastic cancer xenograft model tumor mice were treated with Fab-PE fusion proteins from study day 15 to 33 at dose of 1.0 mg/kg for a total of 7 times (days 15, 17, 19, 22, 24, 26 and 33). Each treatment group consisted of 10 animals. As a result, treatments with the Fab-PE fusion proteins as well as the reference (H8 antibody) showed anti-tumor efficacy with distinct tumor regression of s.c. N87 xenografts ( Figure 16).
  • BxPC3 cells were stably transfected with a plasmid encoding for a luciferase which contains cellular degradation signals (Promega) and has a half-life of about 0.4 h. Single clones were derived and re-assessed for stable plasmid incorporation. Inhibition of protein synthesis by ADP-ribosylation conferred by the pseudomonas exotoxin results in a rapid loss of luciferase and can thus be measured upon cell lysis and incubation with luciferin as loss of signal in comparison to controls.
  • BxPC3/luc in the logarithmic growth phase were detached with Accutase (Sigma) and counted. 40.000 BxPC3/luc cells were seeded per well in 96well-MTP in 100 ⁇ l medium containing 10 % fetal calf serum and were incubated 24 h prior compound addition. TPBG-specific immunoconjugates were added in a concentration range from 3 - 0.0001 nM. All samples were measured in triplicates.
  • An unspecific immunoconjugate has no effect on the luciferase signal (ct). All TPBG-specific immunoconjugates display sub-nM potency. Antibodies of this invention display higher potency than the prior art antibody H8 ( Figure 17A and Table 21).
  • Table 21 Cytotoxic activity mediated by anti-TPBG Fab-PE fusion proteins on human TPBG expressing BxPC-3 cells (EC50 in [nM]) Fab of antibody BxPC-3 X-fold higher potency compared to Fab-PE fusion protein of H8 replicate 1 replicate 2 Unspecific Fab - - H8 0.058 0.020 051 0.027 0.015 2.2 / 1.4 091 0.014 0.007 4.3 / 2.8 097 0.022 0.008 2 . 7 / 2 . 6
  • TPBG-specific Fabs were mixed with anti-human kappa Fab-PE (pseudomonas exotoxin) in a 1:3 molar ratio and kept at room temperature for 15 min to allow for sandwich construct formation. Dilution series of this mixture were prepared in medium. Final concentrations on cells ranged from 133 nM to 0.002 nM as calculated for the TPBG-specific Fab. 24 h post-addition Steady-Glo luciferase assay (Promega) was performed. All samples were measured in triplicates.
  • Affinity of Fab fragments of antibodies 051, 091, and 097 of the invention (as expressed in Example 11) to human TPBG was assessed by BiacoreTM.
  • Fab fragments of prior art antibody H8 were assessed as a control.
  • a CM5 sensor chip was mounted into a Biacore T200 System. Flow cell one was prepared as reference flow cell, by blank immobilization. On flow cell two, about 500 RU of 1.5 ⁇ g/ml huTPBG in pH 4.5 acetate buffer were immobilized (120 s at 10 ⁇ l/min). The amine-coupling was done as described by the manufacturer.
  • the samples were injected in a concentration series of 4.9 nM, 14.8 nM, 44.4 nM, 133.3 nM, 400 nM and 0 nM for 150 s at a flow speed of 50 ⁇ l/min, followed by a dissociation phase of 660 seconds.
  • the sample-antigen-complex was regenerated after every sample injection by one 40 s pulse of Glycine-HCl pH 1.5 at 30 ⁇ l/min and one 40 s long injection of a regeneration solution containing 0.31 M KSCN, 1.22 M MgCl 2 , 0.61 M urea, 1.22 M Gua-HCl and 6.7 mM EDTA at 30 ⁇ /min followed by an extra wash after injection with buffer.
  • Affinity of Fab fragments of antibodies 051, 091, and 097 of the invention (as expressed in Example 11) to minipig TPBG was assessed by BiacoreTM.
  • Fab fragments of prior art antibody H8 were assessed as a control.
  • a CM5 sensor chip was mounted into a Biacore T200 System. Flow cell one was prepared as reference flow cell by blank immobilization. An ⁇ huFab> capture antibody (#28958325, GE-HC) was immobilized via amine coupling onto the flow cells 2, 3 and 4 with an injection time of 1200 s at a concentration of 20 ⁇ g/ml (diluted in the buffer, also provided in the capturing-kit #28958325, GE-HC) and a flow speed of 5 ⁇ l/min.
  • the samples were successively captured on flow cells 2, 3 and 4 with an injection time of 60 s and a flow speed of 20 ⁇ l/min, followed by a 120 seconds long injection of one minipig TPBG dilution out of a dilution series (4.9 nM, 14.8 nM, 44.4 nM, 133.3 nM, 400 nM and 2 ⁇ 0 nM).
  • a dissociation phase of 660 s except the 14.8 nM, 4.9 nM and one 0 nM dilution, for which the diss. time was set to 0 s)
  • the ⁇ huFab> surface was regenerated according to the manufacturer's instructions with the regeneration solution provided in the Fab-capture-kit and the next cycle was carried out.
  • a CM5 sensor chip was mounted into a Biacore T200 System. Flow cell one was prepared as reference flow cell, by blank immobilization. On flow cell two, 80 RU were the aim for the immobilization with 1.5 ⁇ g/ml huTPBG in pH 4.5 acetate buffer. The amine-coupling was done as described by the manufacturer.
  • HBS-EP+ pH 5.5 was used as running and sample buffer
  • HBS-EP+ pH 7.4 was used.
  • the samples were injected in a concentration series of 1.9 nM, 5.6 nM, 2 ⁇ 16.7 nM, 50 nM, 150 nM and 2 ⁇ 0 nM for 300 s at a flow speed of 50 ⁇ l/min.
  • the 50 nM, one 0 nM and one 16.7 nM sample were allowed to dissociate for 1200 s, all other dissociation phases were only 10 s long.
  • the sample-antigen-complex was regenerated after every sample injection by one 40 s pulse of Glycine-HCl pH 1.5 at 30 ⁇ l/min and one 40 s long injection of a regeneration solution containing 0.31 M KSCN, 1.22 M MgCl 2 , 0.61 M urea, 1.22 M Gua-HCl and 6.7 mM EDTA at 30 ⁇ /min followed by an extra wash after injection with buffer.
  • Table 24 Affinity constants of anti-TPBG Fab-PE fusion proteins to human TPBG at different pH values pH Fab comprised in immunoconjugate k a (1/Ms) k d (1/s) K D (M) 5.5 H8 5.43E+04 1.47E-02 2.70E-07 051 3.86E+05 1.13E-04 2.92E-10 091 2.26E+05 2.01E-03 8.89E-09 097 8.08E+05 6.05E-03 7.49E-09 7.4 H8 1.97E+05 7.61E-05 3.85E-10 051 2.86E+05 3.46E-05 1.21E-10 091 2.80E+05 1.29E-04 4.61E-10 097 6.14E+05 1.21E-03 1.97E-09
EP15192002.2A 2015-10-29 2015-10-29 Anticorps anti-tpbg et procédés d'utilisation Ceased EP3184547A1 (fr)

Priority Applications (18)

Application Number Priority Date Filing Date Title
EP15192002.2A EP3184547A1 (fr) 2015-10-29 2015-10-29 Anticorps anti-tpbg et procédés d'utilisation
CN201680062256.2A CN108350083B (zh) 2015-10-29 2016-10-27 抗tpbg抗体及使用方法
JP2018522020A JP7085992B2 (ja) 2015-10-29 2016-10-27 共通軽鎖を有するトランスジェニックウサギ
EP16790315.2A EP3368576A1 (fr) 2015-10-29 2016-10-27 Anticorps anti-tpbg et méthodes d'utilisation
SG10202006332PA SG10202006332PA (en) 2015-10-29 2016-10-27 Transgenic rabbit with common light chain
PCT/EP2016/075882 WO2017072208A1 (fr) 2015-10-29 2016-10-27 Lapin transgénique avec chaîne légère commune
SG11201802698XA SG11201802698XA (en) 2015-10-29 2016-10-27 Transgenic rabbit with common light chain
CN202111178978.3A CN113897371A (zh) 2015-10-29 2016-10-27 具有共同轻链的转基因兔
JP2018522057A JP2019500853A (ja) 2015-10-29 2016-10-27 抗ｔｐｂｇ抗体と使用方法
PCT/EP2016/075881 WO2017072207A1 (fr) 2015-10-29 2016-10-27 Anticorps anti-tpbg et méthodes d'utilisation
CN201680063211.7A CN108347906A (zh) 2015-10-29 2016-10-27 具有共同轻链的转基因兔
EP16790926.6A EP3367786A1 (fr) 2015-10-29 2016-10-27 Lapin transgénique avec chaîne légère commune
CN202111174951.7A CN113862300A (zh) 2015-10-29 2016-10-27 具有共同轻链的转基因兔
US15/965,517 US10434184B2 (en) 2015-10-29 2018-04-27 Anti-TPBG antibodies and methods of use
HK19100672.4A HK1258306A1 (zh) 2015-10-29 2019-01-15 具有共同輕鏈的轉基因兔
HK19101320.8A HK1258850A1 (zh) 2015-10-29 2019-01-25 抗tpbg抗體及使用方法
US16/553,498 US20200108154A1 (en) 2015-10-29 2019-08-28 Anti-tpbg antibodies and methods of use
US18/052,113 US20230332175A1 (en) 2015-10-29 2022-11-02 Transgenic rabbit with common light chain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP15192002.2A EP3184547A1 (fr) 2015-10-29 2015-10-29 Anticorps anti-tpbg et procédés d'utilisation

Publications (1)

Publication Number Publication Date
EP3184547A1 true EP3184547A1 (fr) 2017-06-28

Family

ID=54360972

Family Applications (2)

Application Number Title Priority Date Filing Date
EP15192002.2A Ceased EP3184547A1 (fr) 2015-10-29 2015-10-29 Anticorps anti-tpbg et procédés d'utilisation
EP16790315.2A Pending EP3368576A1 (fr) 2015-10-29 2016-10-27 Anticorps anti-tpbg et méthodes d'utilisation

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP16790315.2A Pending EP3368576A1 (fr) 2015-10-29 2016-10-27 Anticorps anti-tpbg et méthodes d'utilisation

Country Status (6)

Country Link
US (2) US10434184B2 (fr)
EP (2) EP3184547A1 (fr)
JP (1) JP2019500853A (fr)
CN (1) CN108350083B (fr)
HK (1) HK1258850A1 (fr)
WO (1) WO2017072207A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR106188A1 (es) 2015-10-01 2017-12-20 Hoffmann La Roche Anticuerpos anti-cd19 humano humanizados y métodos de utilización
RU2020133262A (ru) 2018-03-12 2022-04-13 Генмаб А/С Антитела
US11608376B2 (en) 2018-12-21 2023-03-21 Hoffmann-La Roche Inc. Tumor-targeted agonistic CD28 antigen binding molecules
US20230257479A1 (en) 2019-09-12 2023-08-17 Genmab A/S Bispecific antibodies binding to 5t4 and cd3 for use in treatment of cancer
KR20210143127A (ko) 2020-05-19 2021-11-26 애니젠 주식회사 신규 뉴클레오린-결합 펩타이드 및 이의 용도

Citations (127)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
WO1988002401A1 (fr) 1986-09-24 1988-04-07 Pastan Ira H Exotoxine de pseudomonas recombinants: construction d'une immunotoxine active avec effets secondaires negligeables
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1990012592A1 (fr) 1989-04-21 1990-11-01 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Proteine recombinante de fusion anticorps-toxine
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1991009949A1 (fr) 1990-01-02 1991-07-11 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Exotoxine de pseudomonase, cytotoxique, recombinee specifique a une cible
WO1991009948A1 (fr) 1990-01-05 1991-07-11 Cornell Research Foundation, Inc. Gene d'actine de riz et promoteur
WO1991009965A1 (fr) 1989-12-21 1991-07-11 The United States Of America, Represented By The Secretary, United States Department Of Commerce Toxine amelioree d'elaboration d'immunotoxines
WO1993001161A1 (fr) 1991-07-11 1993-01-21 Pfizer Limited Procede de preparation d'intermediaires de sertraline
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
WO1993025690A1 (fr) 1992-06-18 1993-12-23 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Exotoxine de pseudomonas recombinee a activite accrue
WO1994011026A2 (fr) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b
WO1994029351A2 (fr) 1993-06-16 1994-12-22 Celltech Limited Anticorps
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
EP0425235B1 (fr) 1989-10-25 1996-09-25 Immunogen Inc Agents cytotoxiques contenant des maytansinoides et leur application thérapeutique
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5608039A (en) 1990-10-12 1997-03-04 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Single chain B3 antibody fusion proteins and their uses
WO1997013529A1 (fr) 1995-10-13 1997-04-17 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Immunotoxine contenant un fragment d'anticorps stabilise au disulfure
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
US5648237A (en) 1991-09-19 1997-07-15 Genentech, Inc. Expression of functional antibody fragments
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5739116A (en) 1994-06-03 1998-04-14 American Cyanamid Company Enediyne derivatives useful for the synthesis of conjugates of methyltrithio antitumor agents
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
WO1998020135A2 (fr) 1996-11-06 1998-05-14 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Proproteines similaires a l'exotoxine de pseudomonas, pouvant etre activees par une protease
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5770710A (en) 1987-10-30 1998-06-23 American Cyanamid Company Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methlytrithio group
US5770701A (en) 1987-10-30 1998-06-23 American Cyanamid Company Process for preparing targeted forms of methyltrithio antitumor agents
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
WO1998055607A2 (fr) 1997-06-04 1998-12-10 Oxford Biomedica (Uk) Limited Vecteur
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
US5869053A (en) 1988-03-04 1999-02-09 Cancer Research Campaign Technology, Ltd. 5T4 antigen from human trophoblasts
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
WO1999051642A1 (fr) 1998-04-02 1999-10-14 Genentech, Inc. Variants d'anticorps et fragments de ceux-ci
EP0972838A1 (fr) 1998-07-15 2000-01-19 Roche Diagnostics GmbH Système de sélection par complémentation d' une auxotrophie en absence d'antibiotique chez E. coli
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000061739A1 (fr) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO2001029246A1 (fr) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un polypeptide
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6291245B1 (en) 1998-07-15 2001-09-18 Roche Diagnostics Gmbh Host-vector system
WO2002031140A1 (fr) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US6630579B2 (en) 1999-12-29 2003-10-07 Immunogen Inc. Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
WO2003085107A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
WO2005035586A1 (fr) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Composition proteique hybride
WO2005035778A1 (fr) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005052006A2 (fr) 2003-11-25 2005-06-09 The Government Of The United States, As Represented By The Secretary Of Health And Human Services Anticorps anti-cd22 et immunoconjugues mutes
WO2005053742A1 (fr) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition a base d'anticorps
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
US20050260186A1 (en) 2003-03-05 2005-11-24 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2006029879A2 (fr) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anticorps anti-ox40l
WO2006031653A2 (fr) 2004-09-10 2006-03-23 Wyeth Anticorps anti-5t4 humanises et conjugues anticorps anti-5t4/calicheamicine
WO2006044908A2 (fr) 2004-10-20 2006-04-27 Genentech, Inc. Formulations d'anticorps
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US20060104968A1 (en) 2003-03-05 2006-05-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
WO2007016150A2 (fr) 2005-07-29 2007-02-08 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH Exotoxines de pseudomonas mutees a antigenicite reduite
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
WO2007031741A1 (fr) 2005-09-14 2007-03-22 Cambridge Antibody Technology Limited Épitopes de cellules t cd4+ exotoxine a de pseudomonas
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
WO2007106744A2 (fr) 2006-03-10 2007-09-20 Wyeth Anticorps anti-5t4 et leurs utilisations
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
US20080069820A1 (en) 2006-08-30 2008-03-20 Genentech, Inc. Multispecific antibodies
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
WO2008077546A1 (fr) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
US7498298B2 (en) 2003-11-06 2009-03-03 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2009032954A1 (fr) 2007-09-04 2009-03-12 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Délétions dans le domaine ii de l'exotoxine a de pseudomonas qui réduisent la toxicité non spécifique
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2009080252A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080254A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080251A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080253A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
WO2010112193A1 (fr) 2009-04-02 2010-10-07 Roche Glycart Ag Anticorps multispécifiques renfermant des anticorps de longueur entière et des fragments fab à chaîne unique
WO2010115589A1 (fr) 2009-04-07 2010-10-14 Roche Glycart Ag Anticorps trivalents bispécifiques
WO2010136172A1 (fr) 2009-05-27 2010-12-02 F. Hoffmann-La Roche Ag Anticorps tri- ou tétraspécifiques
WO2010145793A1 (fr) 2009-06-18 2010-12-23 F. Hoffmann-La Roche Ag Protéines bispécifiques se liant à un antigène tétravalent
WO2010145792A1 (fr) 2009-06-16 2010-12-23 F. Hoffmann-La Roche Ag Protéines bispécifiques se liant à un antigène
WO2011032022A1 (fr) 2009-09-11 2011-03-17 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Exotoxine a pseudomonias améliorée à immunogénicité réduite
WO2012028522A1 (fr) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Charge alcaline
WO2012154530A1 (fr) 2011-05-06 2012-11-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Immunotoxine recombinante ciblant la mésothéline
WO2012170617A1 (fr) 2011-06-09 2012-12-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine de pseudomonas ayant moins d'épitopes immunogènes de lymphocytes t et/ou de lymphocytes b
WO2013040141A1 (fr) 2011-09-16 2013-03-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas des épitopes de cellules b moins immunogènes
WO2015051199A2 (fr) 2013-10-06 2015-04-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas modifiée

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2005319382B2 (en) * 2004-12-21 2011-04-07 Astrazeneca Ab Antibodies directed to angiopoietin-2 and uses thereof
WO2007143168A2 (fr) * 2006-06-02 2007-12-13 Regeneron Pharmaceuticals, Inc. Anticorps dirigés contre le récepteur de l'il-6 humaine, à affinité élevée pour ledit récepteur
WO2010054010A1 (fr) * 2008-11-07 2010-05-14 Fabrus Llc Anticorps anti-dll4 et utilisations associées
JO3246B1 (ar) * 2009-09-09 2018-03-08 Regeneron Pharma اجسام مضادة بشرية ذات ألفة تفاعل عالية مع مستقبل 2 المفعل بالبروتين البشري

Patent Citations (139)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US4892827A (en) 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects
WO1988002401A1 (fr) 1986-09-24 1988-04-07 Pastan Ira H Exotoxine de pseudomonas recombinants: construction d'une immunotoxine active avec effets secondaires negligeables
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5770701A (en) 1987-10-30 1998-06-23 American Cyanamid Company Process for preparing targeted forms of methyltrithio antitumor agents
US5770710A (en) 1987-10-30 1998-06-23 American Cyanamid Company Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methlytrithio group
US5869053A (en) 1988-03-04 1999-02-09 Cancer Research Campaign Technology, Ltd. 5T4 antigen from human trophoblasts
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
WO1990012592A1 (fr) 1989-04-21 1990-11-01 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Proteine recombinante de fusion anticorps-toxine
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5416064A (en) 1989-10-25 1995-05-16 Immunogen, Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
EP0425235B1 (fr) 1989-10-25 1996-09-25 Immunogen Inc Agents cytotoxiques contenant des maytansinoides et leur application thérapeutique
US6417429B1 (en) 1989-10-27 2002-07-09 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
WO1991009965A1 (fr) 1989-12-21 1991-07-11 The United States Of America, Represented By The Secretary, United States Department Of Commerce Toxine amelioree d'elaboration d'immunotoxines
WO1991009949A1 (fr) 1990-01-02 1991-07-11 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Exotoxine de pseudomonase, cytotoxique, recombinee specifique a une cible
WO1991009948A1 (fr) 1990-01-05 1991-07-11 Cornell Research Foundation, Inc. Gene d'actine de riz et promoteur
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5608039A (en) 1990-10-12 1997-03-04 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Single chain B3 antibody fusion proteins and their uses
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993001161A1 (fr) 1991-07-11 1993-01-21 Pfizer Limited Procede de preparation d'intermediaires de sertraline
US5648237A (en) 1991-09-19 1997-07-15 Genentech, Inc. Expression of functional antibody fragments
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
US5602095A (en) 1992-06-18 1997-02-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
WO1993025690A1 (fr) 1992-06-18 1993-12-23 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Exotoxine de pseudomonas recombinee a activite accrue
WO1994011026A2 (fr) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
WO1994029351A2 (fr) 1993-06-16 1994-12-22 Celltech Limited Anticorps
US5767285A (en) 1994-06-03 1998-06-16 American Cyanamid Company Linkers useful for the synthesis of conjugates of methyltrithio antitumor agents
US5877296A (en) 1994-06-03 1999-03-02 American Cyanamid Company Process for preparing conjugates of methyltrithio antitumor agents
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
US5739116A (en) 1994-06-03 1998-04-14 American Cyanamid Company Enediyne derivatives useful for the synthesis of conjugates of methyltrithio antitumor agents
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
WO1997013529A1 (fr) 1995-10-13 1997-04-17 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Immunotoxine contenant un fragment d'anticorps stabilise au disulfure
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
WO1998020135A2 (fr) 1996-11-06 1998-05-14 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Proproteines similaires a l'exotoxine de pseudomonas, pouvant etre activees par une protease
US20030018004A1 (en) 1997-06-04 2003-01-23 Kingsman Susan Mary Vector
WO1998055607A2 (fr) 1997-06-04 1998-12-10 Oxford Biomedica (Uk) Limited Vecteur
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
WO1999051642A1 (fr) 1998-04-02 1999-10-14 Genentech, Inc. Variants d'anticorps et fragments de ceux-ci
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6291245B1 (en) 1998-07-15 2001-09-18 Roche Diagnostics Gmbh Host-vector system
EP0972838A1 (fr) 1998-07-15 2000-01-19 Roche Diagnostics GmbH Système de sélection par complémentation d' une auxotrophie en absence d'antibiotique chez E. coli
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US7332581B2 (en) 1999-01-15 2008-02-19 Genentech, Inc. Polypeptide variants with altered effector function
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2000061739A1 (fr) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
US6420548B1 (en) 1999-10-04 2002-07-16 Medicago Inc. Method for regulating transcription of foreign genes
WO2001029246A1 (fr) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un polypeptide
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US6630579B2 (en) 1999-12-29 2003-10-07 Immunogen Inc. Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
WO2002031140A1 (fr) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
WO2003085107A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
US20050260186A1 (en) 2003-03-05 2005-11-24 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US20060104968A1 (en) 2003-03-05 2006-05-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases
WO2005035586A1 (fr) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Composition proteique hybride
WO2005035778A1 (fr) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
US7498298B2 (en) 2003-11-06 2009-03-03 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2005052006A2 (fr) 2003-11-25 2005-06-09 The Government Of The United States, As Represented By The Secretary Of Health And Human Services Anticorps anti-cd22 et immunoconjugues mutes
WO2005053742A1 (fr) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition a base d'anticorps
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
WO2006031653A2 (fr) 2004-09-10 2006-03-23 Wyeth Anticorps anti-5t4 humanises et conjugues anticorps anti-5t4/calicheamicine
WO2006029879A2 (fr) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anticorps anti-ox40l
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
WO2006044908A2 (fr) 2004-10-20 2006-04-27 Genentech, Inc. Formulations d'anticorps
WO2007016150A2 (fr) 2005-07-29 2007-02-08 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH Exotoxines de pseudomonas mutees a antigenicite reduite
WO2007031741A1 (fr) 2005-09-14 2007-03-22 Cambridge Antibody Technology Limited Épitopes de cellules t cd4+ exotoxine a de pseudomonas
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US8044178B2 (en) 2006-03-10 2011-10-25 Wyeth Llc Anti-5T4 antibodies and uses thereof
WO2007106744A2 (fr) 2006-03-10 2007-09-20 Wyeth Anticorps anti-5t4 et leurs utilisations
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
US20080069820A1 (en) 2006-08-30 2008-03-20 Genentech, Inc. Multispecific antibodies
WO2008077546A1 (fr) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
WO2009032954A1 (fr) 2007-09-04 2009-03-12 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Délétions dans le domaine ii de l'exotoxine a de pseudomonas qui réduisent la toxicité non spécifique
WO2009080252A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080254A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080251A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009080253A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2009089004A1 (fr) 2008-01-07 2009-07-16 Amgen Inc. Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique
WO2010112193A1 (fr) 2009-04-02 2010-10-07 Roche Glycart Ag Anticorps multispécifiques renfermant des anticorps de longueur entière et des fragments fab à chaîne unique
WO2010115589A1 (fr) 2009-04-07 2010-10-14 Roche Glycart Ag Anticorps trivalents bispécifiques
WO2010136172A1 (fr) 2009-05-27 2010-12-02 F. Hoffmann-La Roche Ag Anticorps tri- ou tétraspécifiques
WO2010145792A1 (fr) 2009-06-16 2010-12-23 F. Hoffmann-La Roche Ag Protéines bispécifiques se liant à un antigène
WO2010145793A1 (fr) 2009-06-18 2010-12-23 F. Hoffmann-La Roche Ag Protéines bispécifiques se liant à un antigène tétravalent
WO2011032022A1 (fr) 2009-09-11 2011-03-17 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Exotoxine a pseudomonias améliorée à immunogénicité réduite
WO2012028522A1 (fr) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Charge alcaline
WO2012154530A1 (fr) 2011-05-06 2012-11-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Immunotoxine recombinante ciblant la mésothéline
WO2012170617A1 (fr) 2011-06-09 2012-12-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine de pseudomonas ayant moins d'épitopes immunogènes de lymphocytes t et/ou de lymphocytes b
WO2013040141A1 (fr) 2011-09-16 2013-03-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas des épitopes de cellules b moins immunogènes
WO2015051199A2 (fr) 2013-10-06 2015-04-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Exotoxine a de pseudomonas modifiée

Non-Patent Citations (141)

* Cited by examiner, † Cited by third party
Title
ALEWINE C ET AL., MOL CANCER THER., 2014, pages 2653 - 61
ALEWINE ET AL., MOL. CANCER THER., vol. 13, no. 11, 2014, pages 2653 - 61
ALMAGRO, J.C.; FRANSSON, J., FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633
BACA, M. ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684
BOERNER, P. ET AL., J. IMMUNOL., vol. 147, 1991, pages 86 - 95
BRAVO ET AL., J BIOL CHEM, vol. 269, no. 14, 1994, pages 25830 - 25837
BRENNAN, M. ET AL., SCIENCE, vol. 229, 1985, pages 81 - 83
BRODEUR, B.R. ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
BRUGGEMANN, M. ET AL., J. EXP. MED., vol. 166, 1987, pages 1351 - 1361
CARTER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285 - 4289
CHARI, R.V. ET AL., CANCER RES., vol. 52, 1992, pages 127 - 131
CHARLTON, K.A.: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 245 - 254
CHAUDHARY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1991, pages 308 - 312
CHEN, Y. ET AL., J. MOL. BIOL., vol. 293, 1999, pages 865 - 881
CHENG ET AL., J. CLIN. ONCOL., vol. 22, no. 4, 2004, pages 602 - 9
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOWDHURY, P.S., METHODS MOL. BIOL., vol. 207, 2008, pages 179 - 196
CLACKSON, T. ET AL., NATURE, vol. 352, 1991, pages 624 - 628
CLIN. CANCER RES., vol. 11, no. 10, 2005, pages 3814 - 20
CLYNES, R. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656
CRAGG, M.S. ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052
CRAGG, M.S.; M.J. GLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743
CUNNINGHAM, B.C; WELLS, J.A., SCIENCE, vol. 244, 1989, pages 1081 - 1085
DALL'ACQUA, W.F. ET AL., METHODS, vol. 36, 2005, pages 43 - 60
DUBOWCHIK, G.M. ET AL., BIOORG. & MED. CHEM. LETTERS, vol. 12, 2002, pages 1529 - 1532
DUCKERT ET AL., PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 17, no. 1, 2004, pages 107 - 112
DUNCAN, A.R.; WINTER, G., NATURE, vol. 322, 1988, pages 738 - 740
EDELMAN, G.M. ET AL., PNAS, vol. 63, 1969, pages 78 - 85
FELLOUSE, F.A., PROC. NATL. ACAD. SCI. USA, vol. 101, 2004, pages 12467 - 12472
FLATMAN, S. ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87
GAZZANO-SANTORO, H. ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163 - 171
GERNGROSS, T.U., NAT. BIOTECH, vol. 22, 2004, pages 1409 - 1414
GRAHAM, F.L ET AL., J. GEN VIROL, vol. 36, 1977, pages 59 - 74
GRIFFITHS, A.D. ET AL., EMBO J., vol. 12, 1993, pages 725 - 734
GRUBER, M ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368 - 5374
GUYER, R.L. ET AL., J. IMMUNOL., vol. 117, 1976, pages 587 - 593
HANSEN ET AL., JOURNAL OF IMMUNOTHERAPY, vol. 33, no. 3, 2010, pages 297 - 304
HARLOW, E.; LANE, D.: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY, article "Chapter 14,"
HATSUZAWA ET AL., J BIOL CHEM, vol. 267, 1992, pages 16094 - 16099
HELLSTROM, I. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 1499 - 1502
HELLSTROM, I. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 7059 - 7063
HINMAN, L.M ET AL., CANCER RES., vol. 53, 1993, pages 3336 - 3342
HOLLIGER, P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HOOGENBOOM, H.R. ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 178, 2001, pages 1 - 37
HOOGENBOOM, H.R. ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 178, 2002, pages 1 - 37
HOOGENBOOM, H.R.; WINTER, G., J. MOL. BIOL., vol. 227, 1992, pages 381 - 388
HUDSON, P.J ET AL., NAT. MED, vol. 9, 2003, pages 129 - 134
HUDSON, P.J. ET AL., NAT. MED, vol. 9, 2003, pages 129 - 134
HUDSON, P.J. ET AL., NAT. MED., vol. 9, 2003, pages 129 - 134
HWANG ET AL., CELL, vol. 48, 1987, pages 129 - 136
IDUSOGIE, E.E. ET AL., J. IMMUNOL., vol. 164, 2000, pages 4178 - 4184
JEFFREY, S.C. ET AL., BIOORG. MED. CHEM, vol. 16, 2006, pages 358 - 362
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest, 5th ed.,", 1991, NIH PUBLICATION 91-3242
KAM, N.W ET AL., PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 11600 - 11605
KANDA, Y. ET AL., BIOTECHNOL. BIOENG., vol. 94, 2006, pages 680 - 688
KASHMIRI, S.V. ET AL., METHODS, vol. 36, 2005, pages 25 - 34
KIM, J.K. ET AL., J. IMMUNOL., vol. 24, 1994, pages 2429 - 2434
KINDT, T.J. ET AL.: "Kuby Immunology, 6th ed.,", 2007, W.H. FREEMAN AND CO., pages: 91
KING, H.D. ET AL., J. MED. CHEM., vol. 45, 2002, pages 4336 - 4343
KLIMKA, A. ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260
KONDO ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 9470 - 9475
KOSTELNY, S.A ET AL., J. IMMUNOL., vol. 148, 1992, pages 1547 - 1553
KOZBOR, D., J. IMMUNOL., vol. 133, 1984, pages 3001 - 3005
KRATZ, F. ET AL., CURR. MED. CHEM., vol. 13, 2006, pages 477 - 523
KREITMAN ET AL., CLIN CANCER RES., vol. 15, 2009, pages 5274 - 5279
KREITMAN ET AL., J CLIN ONCOL., vol. 18, 2000, pages 1622 - 1636
KREITMAN ET AL., J. CLIN. ONCOL., vol. 30, no. 15, 2012, pages 1822 - 8
LEE, C.V. ET AL., J. IMMUNOL. METHODS, vol. 284, 2004, pages 119 - 132
LEE, C.V. ET AL., J. MOL. BIOL., vol. 340, 2004, pages 1073 - 1093
LI, H. ET AL., NAT. BIOTECH, vol. 24, 2006, pages 210 - 215
LI, J. ET AL., PROC. NAT]. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562
LIU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 109, no. 29, 2012, pages 11782 - 7
LODE, H.N. ET AL., CANCER RES., vol. 58, 1998, pages 2925 - 2928
LONBERG, N., CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 450 - 459
LONBERG, N., NAT. BIOTECH, vol. 23, 2005, pages 1117 - 1125
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MARKS, J.D. ET AL., J. MOL. BIOL., vol. 222, 1992, pages 581 - 597
MARKS, J.D.; BRADBURY, A., METHODS IN MOLECULAR BIOLOGY, vol. 248, 2003, pages 161 - 175
MATHER, J.P ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, J.P, BIOL. REPROD., vol. 23, 1980, pages 243 - 252
MAZOR ET AL., PROC. NATL. ACAD. SCI. USA, vol. 111, no. 23, 2014, pages 8571 - 8576
MAZOR ET AL., PROC. NATL. ACAD. SCI. USA, vol. 111, no. 23, pages 8571 - 8576
MAZOR R ET AL., PNAS, vol. 111, 2014, pages 8571 - 8576
MCCAFFERTY, J. ET AL., NATURE, vol. 348, 1990, pages 552 - 554
MILSTEIN, C.; CUELLO, A.C, NATURE, vol. 305, 1983, pages 537 - 540
MOLLOY ET AL., J BIOL CHEM, vol. 267, 1992, pages 16396 - 16402
MORRIS, G.E.: "Epitope Mapping Protocols, In: Methods in Molecular Biology", vol. 66, 1996, HUMANA PRESS
MORRISON, S.L. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MYERS ET AL., CANCER GENE THER., vol. 9, 2002, pages 884 - 896
MZ LI ET AL., NATURE METHODS, vol. 4, 2007, pages 251 - 256
NAGY, A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 97, 2000, pages 829 - 834
NI, J., XIANDAI MIANYIXUE, vol. 26, 2006, pages 265 - 268
OKAZAKI, A. ET AL., J. MOL. BIOL., vol. 336, 2004, pages 1239 - 1249
ONDA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 105, no. 32, 2008, pages 11311 - 6
ONDA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 108, no. 14, 2011, pages 5742 - 7
OSBOURN, J. ET AL., METHODS, vol. 36, 2005, pages 61 - 68
OSOL, A.: "Remington's Pharmaceutical Sciences, 16th edition,", 1980
PADLAN, E.A., MOL. IMMUNOL., vol. 28, 1991, pages 489 - 498
PAI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 3358 - 62
PASTAN ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1333, 1997, pages C1 - C6
PASTAN ET AL., LEUKEMIA & LYMPHOMA, vol. 52, no. 2, 2011, pages 87 - 90
PETKOVA, S.B ET AL., INT. IMMUNOL., vol. 18, 2006, pages 1759 - 1769
PLUECKTHUN, A.: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315
PORTOLANO, S. ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887
PRESTA, L.G ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 2632
PRESTA, L.G. ET AL., CANCER RES., vol. 57, 1997, pages 4593 - 4599
QUEEN, C. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033
RAVETCH, J.V.; KINET, J.P., ANNU. REV. IMMUNOL., vol. 9, 1991, pages 457 - 492
RIECHMANN, I. ET AL., NATURE, vol. 332, 1988, pages 323 - 329
RIPKA, J. ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 249, 1986, pages 533 - 545
ROCKWELL; THORNER, TRENDS BIOCHEM. SCI, vol. 29, 2004, pages 80 - 87
ROSCOE ET AL., INFECTION AND IMMUNITY, vol. 62, no. 11, 1994, pages 5055 - 65
ROSOK, M.J. ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618
RS HAUN ET AL., BIOTECHNIQUES, vol. 13, 1992, pages 515 - 518
SAMBROOK, J. ET AL.: "Molecular cloning: A laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAPRA ET AL., MOL CANE THER, vol. 12, 2013, pages 38 - 47
SEETHARAM ET AL., J. BIOL. CHEM., vol. 266, 1991, pages 17376 - 17381
SHAW ET AL., BIOCHEM. J., vol. 363, 2002, pages 137 - 45
SHAW ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1524, 2000, pages 238 - 246
SHIELDS, R.L. ET AL., J. BIOL. CHEM., vol. 276, 2001, pages 6591 - 6604
SIDHU, S.S. ET AL., J. MOL. BIOL., vol. 338, 2004, pages 299 - 310
SIMS, M.J. ET AL., J. IMMUNOL., vol. 151, 1993, pages 2296 - 2308
THOMAS, NAT REV MOL CELL BIOL, vol. 10, 2002, pages 753 - 66
THOMAS, NAT. REV. MOL. CELL BIOL, vol. 3, 2002, pages 753 - 766
TIM EISEN ET AL: "Naptumomab Estafenatox: Targeted Immunotherapy with a Novel Immunotoxin", CURRENT ONCOLOGY REPORTS, vol. 16, no. 2, 1 February 2014 (2014-02-01), GB, XP055251158, ISSN: 1523-3790, DOI: 10.1007/s11912-013-0370-0 *
TORGOV, M.Y. ET AL., BIOCONJUG. CHEM., vol. 16, 2005, pages 717 - 721
TRAUNECKER, A ET AL., EMBO J., vol. 10, 1991, pages 3655 - 3659
TUTT, A. ET AL., J. IMMUNOL., vol. 147, 1991, pages 60 - 69
URLAUB, G. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 4220
VAN DIJK, M.A.; VAN DE WINKEL, J.G., CURR. OPIN. PHARMACOL., vol. 5, 2001, pages 368 - 374
VITETTA, E.S. ET AL., SCIENCE, vol. 238, 1987, pages 1098 - 1104
VOLLMERS, H.P.; BRANDLEIN, S., HISTOLOGY AND HISTOPATHOLOGY, vol. 20, 2005, pages 927 - 937
VOLLMERS, H.P; BRANDLEIN, S., METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, 2005, pages 185 - 191
WELDON ET AL., BLOOD, vol. 113, 2009, pages 3792 - 3800
WELDON; PASTAN, FEBS J, vol. 278, no. 23, 2011, pages 4683 - 4700
WINTER, G. ET AL., ANN. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455
WOODS ET AL., BIOCHEM. J., vol. 366, 2002, pages 353 - 65
WRIGHT, A.; MORRISON, S.L., TIBTECH, vol. 15, 1997, pages 26 - 32
YAMANE-OHNUKI, N. ET AL., BIOTECH. BIOENG, vol. 87, 2004, pages 614 - 622
YAMANE-OHNUKI, N. ET AL.: "Biotech. Bioeng.", BIOTECH. BIOENG., vol. 87, 2004, pages 614 - 622
YAZAKI, P.; WU, A.M: "Methods in Molecular Biology", vol. 248, 2004, HUMANA PRESS, pages: 255 - 268

Also Published As

Publication number Publication date
CN108350083B (zh) 2021-11-26
EP3368576A1 (fr) 2018-09-05
US10434184B2 (en) 2019-10-08
CN108350083A (zh) 2018-07-31
US20200108154A1 (en) 2020-04-09
HK1258850A1 (zh) 2019-11-22
US20180311371A1 (en) 2018-11-01
WO2017072207A1 (fr) 2017-05-04
JP2019500853A (ja) 2019-01-17

Similar Documents

Publication Publication Date Title
US20220363755A1 (en) Anti-tim3 antibodies and methods of use
EP2747781B1 (fr) Anticorps bispécifiques spécifiques pour les antigènes d'activation des lymphocytes t et un antigène tumoral et procédés d'utiliation correspondants
EP3143049B1 (fr) Anticorps bispécifiques her3/her2 se liant à la structure en épingle à cheveux béta de her3 et au domaine ii de her2
US10434184B2 (en) Anti-TPBG antibodies and methods of use
EP2917243B1 (fr) Anticorps her3 se liant à la structure en épingle à cheveux bêta de her3
KR102193080B1 (ko) 항-jagged 항체 및 사용 방법
EP2917242B1 (fr) Anticorps anti-her3/her4 se liant à la structure en épingle à cheveux bêta de her3 et de her4
EP3143048B1 (fr) Anticorps her3 se liant à la structure en épingle à cheveux bêta de her3
JP2018520658A (ja) ヒト化抗エボラウイルス糖タンパク質抗体及びその使用
US20180100022A1 (en) Her1 antigen binding proteins binding to the beta-hairpin of her1
US10745470B2 (en) Modified anti-tenascin antibodies and methods of use

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20170720